MXPA01008108A

MXPA01008108A - USE OF RECOMBINANT PARAINFLUENZA VIRUSES (PIVs) AS VECTORS TO PROTECT AGAINST INFECTION AND DISEASE CAUSED BY PIV AND OTHER HUMAN PATHOGENS.

Info

Publication number: MXPA01008108A
Application number: MXPA01008108A
Authority: MX
Inventors: C Schmidt Alexander
Original assignee: Health
Priority date: 1999-12-10
Filing date: 2000-12-08
Publication date: 2004-11-12
Also published as: EP1179054A2; WO2001042445A3; CA2362685A1; WO2001042445A2; CN102766605A; JP4795597B2; AU2073101A; JP2003516148A; AU785148B2; IL144831A0; CN1347453A

Abstract

Chimeric parainfluenza viruses (PIVs) are provided that incorporate a PIV vector genome or antigenome and one or more antigenic determinant(s) of a heterologous PIV or non PIV pathogen. These chimeric viruses are infectious and attenuated in humans and other mammals and are useful in vaccine formulations for eliciting an immune responses against one or more PIVs, or against a PIV and non PIV pathogen. Also provided are isolated polynucleotide molecules and vectors incorporating a chimeric PIV genome or antigenome which includes a partial or complete PIV vector genome or antigenome combined or integrated with one or more heterologous gene(s) or genome segment(s) encoding antigenic determinant(s) of a heterologous PIV or non PIV pathogen. In preferred aspects of the invention, chimeric PIV incorporate a partial or complete human, bovine, or human bovine chimeric, PIV vector genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a heterologous PIV or non PIV pathogen, wherein the chimeric virus is attenuated for use as a vaccine agent by any of a variety of mutations and nucleotide modifications introduced into the chimeric genome or antigenome.

Description

USE OF RECOMBINANT PARINFLUENZA VIRUSES (PIVs) AS VECTORS FOR THE PROTECTION AGAINST INFECTION AND DISEASE CAUSED BY PIV AND OTHER HUMAN PATHOGENS BACKGROUND OF THE INVENTION The human parainfluenza type 3 virus (HPIV3) is a common cause of serious lower respiratory tract infections in infants and children less than one year of age. It is the second only for respiratory syncytial virus (RSV) as a cause that leads to hospitalization for viral lower respiratory tract disease in this age group (Collins et al., P.1220-1243 in BN Fields (Knipe et al., eds.), Fields Virology, 3rd ed., Vol 1. Lippincott-Raven Publishers, Philadelphia, 1996 / Cro et al., Vaccine 13: 415-421, 1995; et al., J. Infect. Dis. 176: 1423-1427, 1997). Infections with this virus result in substantial morbidity in children younger than 3 years of age. HPIV1 and HPIV2 are the main etiological agents of laryngotracheobronchitis (croup) and can also cause pneumonia and severe bronchitis (Collins et al., 3rd ed., In "Fields Virology," BN Fields, DM Knipe, PM Howley, RM Chanock, JL Melnick , TP Monath, B. Roizrnan, and SE Straus, Eds. , Vol. 1, pp. 1205-1243. Lippincott-Raven Publishers, Philadelphia, 1996). In a long-term study over a period of 20 years, HPIV1, HPIV2 and HPIV3 were identified as etiologic agents for 6.0%, 3.2% and 11.5%, respectively, of hospitalizations for respiratory tract disease taking into account in total for the 18% of hospitalizations and, for this reason, there is a need for an effective vaccine (urphy et al., Virus Res. 11: 1-15, 1988). Parainfluenza viruses have been identified in a significant proportion of cases of virally induced middle ear effusions in children with otitis media (Heikkinen et al., N. Engl. J. ed. 340: 260-4 / 1999). Thus, there is a need to produce a vaccine against these viruses that can prevent the severe disease of the lower respiratory tract and the otitis media that accompanies these HPIV infections. HPIV1, HPIV2 and HPIV3 are distinct serotypes that do not produce significant cross-protective immunity. Despite considerable efforts to develop effective HPIV vaccine therapies, there are no approved vaccine agents that have been achieved for any HPIV serotype, nor to improve conditions related to HPIV. To date, only two candidates for live attenuated PIV vaccine have received particular attention. One of these candidates is a bovine PIV strain (BPIV3) that is antigenically related to HPIV3 and that has been shown to protect animals against HPIV3. BPIV3 is attenuated, genetically stable and immunogenic in infants and children (Karron et al., J. Inf. Dis. 171: 1107-14, 1995a; Karron et al., J. Inf. Dis. 172; 1445-1450, 1995b ). A second candidate for PIV3 vaccine JS cp45 is a cold-adapted mutant of the wild-type strain (ts) JS of HPIV3 (Karron et al., J, Inf. Dis 172: 1445-1450, 1995b; Belshe et al. ., Med. Virol. 10: 235-42, 1982). This candidate for cold-pass PIV3 vaccine, attenuated in vivo exhibits temperature-sensitive phenotypes (st), with adaptation to cold (ac) and attenuation (att) that are stable after viral replication in vivo. The cp45 virus provides protection against the inoculation of human PIV3 in experimental animals and is attenuated, genetically stable and immunogenic in infants and seronegative human children (Hall et al., Virus Res. 22: 173-184, 1992; Karron et al. , J. 'Inf. Dis. 172: 1445-1450, 1995b). Most promising prospects to date are live attenuated vaccine viruses because they have been shown to be effective in non-human primates even in the presence of passively transferred antibodies, an experimental situation that simulates that it occurs in very young infants who have acquired antibodies. through the maternal route (Crowe et al., Vaccine 13: 847-855, 1995; Durbin et al., J. Infect. Dis, 179; 1345-1351, 1999). Two candidates for live-attenuated PIV3 vaccine, a derivative of the wild type PIV3 JS strain (designated PIV3cp45) sensitive to temperature (ts) and a bovine PIV3 strain (BPIV3), are subjected to clinical evaluation (Karron et al., Pediatr Infect, Dis. J. 15: 650-654, 1996, Karron et al., J. Infect. Dis. 171: 1107-1114, 1995a, Karron et al., J. Infect. Dis. 172, 1445- 1450, 1995b). The candidate for live attenuated PIV3cp45 vaccine was derived from the JS strain of HPIV3 via the serial passage in cell culture at low temperature and has been found to protect against inoculation by HPIV3 in experimental animals and which is satisfactorily attenuated, is genetically stable and immunogenic in infants and seronegative human children (Belshe et al, J. Med. Virol., 10: 235-242, 1982; Belshe et al., Infect. Immun., 37: 160-5, 1982; Clements et al., J. Clin. Microbiol., 29: 1175-82, 1991; Crookshanks et al., J. Med. J. Virol., 13: 243-9, 1984; Hall et al., Virus Res. 22: 173-184, 1992; et al., J. Infect. Dis. 172: 1445-1450, 1995b). Because these viruses for candidate vaccine for PIV3 are biologically derived, there are no proven methods to adjust the level of attenuation that should be necessary from continuous clinical trials.

To facilitate the development of candidates for PIV vaccine, recombinant DNA technology has recently made it possible to recover infectious, negative-strand cDNA RNA viruses (for review, see Conzelmann, J. Gen. Virol. 7_: 381-89 , 1996; Palese et al., Proc. Nati, Acad. Sci. USA 93: 11354-58, 1996). In this context, recombinant rescue has been reported for infectious respiratory syncytial virus (RSV), rabies virus (RaV, simian virus 5 (SV5), rinderpest virus, Ne castle disease virus (NDV), vesicular stomatitis virus (VSV), measles virus (MeV) and Linea virus (SeV, its acronyms in English) from antigenomic RNA encoded by cDNA in the presence of essential viral proteins (see, for example, Garcin et al., EMBO J. 14: 6087-6094, 1995, Lawson et al., Proc. Nati. Acad. Sci. USA 92: 4477-81, 1995; Radecke et al., EMBO J. 14: 5773-5784, 1995; Schnell et al., EMBO J. 13: 4195-203, 1994; Helan et al., Proc. Nati, Acad Sci, USA 92: 8388-92, 1995, Hoffman et al., J. Virol. 71: 4272-4277, 1997, Kato et al., Genes to Cells 1: 569-579, 1996, Roberts. et al., Virology 247: 1-6, 1998; Baron et al., J Virol. 7_1: 1265-1271, 1997; International Publication No. O 97/06270; Collins et al., Proc. Nati Acad. Sci.

USES. 92: 11563-11567, 1995; U.S. Patent Application No. 08 / 892,403, filed July 15, 1997 (corresponding to published International Application No. WO 98/02530 and to the priority of U.S. Provisional U.S. Nos. 60 / 047,634 , filed on May 23, 1997, 60 / 046,141, filed May 9, 1997 and 60/021, 773, filed July 15, 1996); U.S. Patent Application No. 09 / 291,894, filed April 13, 1999; International Application No. PCT / USOO / 09695, filed April 12, 2000 (which claims the priority of United States Provisional Patent Application Serial No. 60 / 129,006, filed April 13, 1999); International Application No. PCT / USOO / 17755, filed June 23, 2000 (which claims the priority of United States Provisional Patent Application Serial No. 60 / 143,132, filed by Bucholz et al. July 1999); Juhasz et al., J. Virol. 71: 5814-5819, 1997; He et al. Virology 237: 249-260, 1997; Peters et al. J. Virol. 73: 5001-5009, 1999; Barón et al. J. Virol. 71: 1265-1271, 1997; Whitehead et al., Virology 247: 232-9, 1998a; Whitehead et al., J. Virol. 72: 4467-4471, 1998b; Jin et al. Virology 251: 206-214, 1998; Bucholz et al. J. Virol. 73: 251-259, 1999; and Whitehead et al., J. Virol. 73: 3438-3442, 1999, each incorporated herein by reference in its entirety for all purposes). More specifically referring to the current invention, a method has recently been developed for producing HPIV with a wild-type cDNA phenotype for the recovery of the infectious, recombinant HPIV3 JS strain (see, for example, Durbin et al., Virology. 235: 323-332, 1997; United States Patent Application Serial No. 09/083, 793, filed May 22, 1998 (corresponding to United States Provisional Application No. 60 / 059,385, filed) on September 19, 1997), and United States Provisional Application No. 60 / 047,575, filed May 23, 1997 (corresponding to International Publication No. 0 98153078), each incorporated herein by reference ). In addition, these exposures allow the genetic manipulation of viral cDNA clones to determine the genetic basis of phenotypic changes in biological mutants, for example, whose mutations in HPV3 cp45 virus specify their ts, ca, att phenotypes, and whose genes or Genomic segments of BPIV3 specify its attenuation phenotype. Additionally, these and related exposures make it feasible to construct novel PIV vaccine candidates having a wide range of different mutations and to assess their level of attenuation, immunogenicity, and phenotypic stability (see also, U.S. Application No. 09 / 586,479, filed on June 1, 2000 (corresponding to Provisional Patent Application Serial No. 60 / 143,134, filed July 9, 1999); and 09 / 350,821, filed by Durbin et al. on July 9, 1999, each incorporated herein by reference). In this way, PIV3, (r) PIV3, recombinant, wild-type, infectious, as well as several of the ts derivatives, have now been recovered from cDNA and inverse genetic systems have been used to generate infectious viruses carrying defined attenuating mutations and to study the genetic basis of attenuation of existing vaccine viruses. For example, the three amino acid substitutions found in the L-gene of cp45, individually or in combination, have been found to specify the ts and attenuation phenotypes. Additional ts and attenuation mutations are present in other regions of PIV3cp45. In addition, a candidate for chimeric PIV1 vaccine has been generated using the PIV3 cDNA rescue system by replacing the open reading frames (ORFs) HN and F of PIV3 with those of PIV1 in a length cDNA. complete PIV3 containing the three attenuating mutations in L. The recombinant chimeric virus derived from this cDNA is dested PIV3r-l.cp45L (Skiadopoulos et al., J. Virol. 72: 1762-8, 1998; Tao et al., J Virol 72: 2955-2961, 1998; Tao et al., Vaccine 17: 1100-1108, 1999, incorporated herein by reference). PIV3r-l. cp45L was attenuated in hamsters and a high level of resistance was induced to inoculate with PIV1. Still another recombinant chimeric virus, dested PIV3r-1, has been produced. cp45, which contains 12 of the 15 cp45 mutations, that is, excluding the mutations that occur in HN and F. This candidate for recombinant vaccine is highly attenuated in the upper and lower respiratory tract of hamsters and induces a high level of protection against HPIV1 infection (Skiadopoulos et al., Vaccine 18: 503-510, 1999). Recently, several studies have focused on the possible use of viral vectors to express foreantigens towards the goal of developing vaccines against a pathogen for which other vaccine alternatives have not been successfully tested. In this context, several reports suggest that foregenes can be successfully inserted into a genome or antigenome of viruses with recombinant negative-strand RNA, with varying effects (Bukreyev et al., J. Virol. 70: 6634-41, 1996; Bukreyev et al., Proc. Nati. Acad Sci. USA 96 ^: 23 67-72, 1999; Finke et al., J. Virol. 71: 7281-8, 1997; Hasan et al., J. Gen. Virol 78: 2813-20, 1997, He et al., Virology 237: 249-60, 1997, Jin et al., Virology 251: 206-14, 1998, Johnson et al., J. Virol. 71 ^: 5060-8, 1997; Kahn et al., Virology 254: 81-91, 1999; Kretzschmar et al., J. Virol. 71: 5982-9, 1997; Mebatsion et al., Proc. Nati Acad. Sci. U.S.A. 93 ^: 7310-4, 1996; Moriya et al., FEBS Lett. 425: 105-11, 1998; Roberts et al., J.'virol. 73: 3723-32, 1999; Roberts et al., J. Virol. 72: 4704-11, 1998; Roberts et al., Virolo 247: 1-6, 1998; Sakai et al., FEBS Lett. 456: 221-226, 1999; Schnell et al. , Proc. Natl. Acad. Sci. U.S.A. 93: 11359-65, 1996a; Schnell et al., J. Virol. 70: 2318-23, 1996b; Schnell et al., Cell 90 ^: 849-57, 1997; Singh et al. , J. Gen. Virol. 80: 101-6, 1999; Singh et al., J. Virol. 73: 4823-8, 1999; Spielhofer et al., J. Virol. 72: 2150-9, 1998; Yu et al., Genes to Cells 2: 457-66 et al., 1999; U.S. Patent Application Serial No. 09/614, 285, filed July 12, 2000 (corresponding to U.S. Provisional Patent Application Serial No. 60 / 143,425, filed July 13 of 1999, each incorporated herein by reference). When inserted into the viral genome under the control of the initial gene signals and the final viral transcription gene, the foreign gene can be transcribed as a separate mRNA and provides significant protein expression. Surprisingly, in some cases the foreign sequence has been reported to be stable and capable of expressing a functional protein during several in vitro passages.

However, in order to successfully develop vectors for the use of the vaccine, it is insufficient to simply demonstrate a high stable level of protein expression. For example, this has been possible since before the mid-1980s with recombinant vaccinia viruses and adenoviruses, and even these vectors have proven to be disappointing in the development of vaccines for human use. Similarly, most of the unsegmented negative chain viruses that have been developed as vectors and do not possess immunization properties or strategies arranged for human use. Examples in this context include vesicular stomatitis virus, an ungulate pathogen with no history of administration to humans except for some laboratory accidents; the Líneai virus, a mouse pathogen with no history of administration to humans; simian virus 5, a canine pathogen with no history of administration to humans; and an attenuated strain of measles virus that must be administered systemically and could be neutralized by measles-specific antibodies present in almost all humans due to maternal antibodies and the widespread use of an authorized vaccine. In addition, some of these previous vector candidates have adverse effects, such as, for example, immunosuppression, which are directly inconsistent with their use as vectors. In this way, vectors whose growth characteristics, tropisms and other biological properties make them suitable as vectors for use in humans must be identified. In addition, it is necessary to develop a viable vaccination strategy that includes an immunogenic and effective route of administration. The three human mononegaviruses that have been currently considered as vectors for the vaccine, namely the measles, mumps and rabies viruses, have additional limitations that combine to make them weak candidates for further development as vectors. For example, measles virus has been considered to utilize a vector for the hepatitis B virus protective antigen (Singh et al., J. Virol. 73: 4823-8, 1999). However, only this combined measles-hepatitis B virus vaccine, similar to the approved measles virus vaccine, could be provided after nine months of age, while the current hepatitis B virus vaccine is recommended for use in early childhood. This is because the currently licensed measles virus vaccine is administered parenterally and is very sensitive to neutralization and immunosuppression by maternal antibodies and therefore is not effective if administered before 9 to 15 months of age . Thus, could not be used for vector antigens that cause disease in early childhood and therefore may not be useful for viruses such as for example SV and HPIV. Another well-known characteristic effect of measles virus infection is the immunosuppression provided by the virus, which may last for several months. L immunosuppression may not be a desirable feature for a vector. The vaccine for the attenuated measles virus is associated with altered immune responses and excess mortality when administered at an increased dose, which could be due at least in part to the immunosuppression induced by the virus and indicates that even a virus of Dimmed measles may not be suitable as a vector. In addition, the use of measles virus as a vector could be inconsistent with the global effort to eradicate this pathogen. Indeed, for these reasons it would be desirable to terminate the use of live measles virus and replace the vaccine for the measles virus present with a PIV vector expressing protective antigens of the measles virus, as described herein. Rabies virus, a rare cause of human infection, has been considered to be used as a vector (Mebatsion et al., Proc. Nati. Acad. Sci. USA 93; 7310-4, 1996), although it is unlikely that a vector that is 100% fatal to humans could be developed to be used as a vector of attenuated virus in vivo, especially due to immunity to rabies viruses, other than A ubiquitous human pathogen is not needed for the general population. While mumps and measles viruses are less pathogenic, infection by any virus can involve undesirable characteristics. Mumps virus infects the parotid gland and can spread to the testicles, sometimes resulting in infertility. The measles virus establishes a viremia and the widespread nature of this infection is exemplified by the associated widespread rash. Mild encephalitis during mumps and measles infection is not uncommon. The measles virus is also associated with a rare progressive fatal neurological disease called subacute sclerosing encephalitis. In contrast, PIV infection and disease in normal individuals is limited to the respiratory tract, a site that is much more advantageous for immunization than the parental route. Viremia and its spread to secondary sites can occur in severely immunocompromised experimental animals and humans, although this is not a characteristic of typical PIV infection. Acute respiratory tract disease is the only disease associated with IVP. In this way, the use of PIV as vectors will avoid complications such as for example the interaction of the virus with peripheral lymphocytes on the basis of their biological characteristics, leading to immunosuppression, or infection of secondary organs such as for example the testes or the central nervous system, leading to other complications. Among the host pathogens of humans for which a vector-based vaccine approach may be desirable is the measles virus. A live attenuated vaccine has been available for more than three decades and has been quite successful in eradicating measles disease in the United States. However, the World Health Organization estimates that more than 45 million cases of measles still occur annually, particularly in developing countries and the virus contributes to approximately one million deaths per year. The measles virus is a member of the genus Morbillivirus of the family Paramyxoviridae (Griffin et al., In "Fields Virology", BN Fields, DM Knipe, PM Howley, RM Chanock, JL Melnick, TP Monath, B. Roizman, and SE Straus, Eds., Vol. 1, pp. 1267-1312, Lippincott-Raven Publishers, Philadelphia, 1996). It is one of the most infectious and contagious agents known to man and transmitted from person to person through the respiratory tract (Griffin et al., In "Fields Virology", BN Fields, DM Knipe, PM Howley, RM C anock, JL Melnick, TP Monath, B. Roizman, and SE Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The measles virus has a complex pathogenesis, which involves replication both in the respiratory tract and in the various systemic sites (Griffin et al., In "Fields Virology", BN Fields, DM Knipe, PM Howley, RM Chanock, JL Melnick , TP Monath, B. Roizinan, and SE Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Although both virus-specific IgA mucosal and measles IgG antibodies in serum may be involved in the control of measles virus, the absence of measles virus disease in very young infants who possess measles-specific antibodies acquired via maternal serum antibodies as a principal mediator of disease resistance (Griffin et al., In "Fields Virology", BN Fields, DM Knipe, PM Howley, Chanock RM, Melnick JL, TP Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, p. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The two measles virus glycoproteins, hemagglutinin (HA) and fusion (F) proteins, are the main neutralizing and protective antigens (Griffin et al., In "Fields Virology", BN Fields, DM Knipe, PM Ho law, RM Chanock, Melnick JL, Monath TP, Roizrnan B., and SE Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Currently available live attenuated measles vaccine is administered by a parenteral route (Griffin et al., In "Fields Virology", BN Fields, DM Knipe, PM Howley, RM Chanock, JL Melhick, TP Monath, B. Roizrnan, and S. E Straus, Eds., Vol. 1, p.1267-1312, Lippincott-Raven Publishers, Philadelphia, 1996). Both the wild-type measles virus and the vaccine virus are very easy to neutralize by antibodies and the measles virus vaccine becomes non-infectious even at very low levels of measles-specific neutralizing antibodies acquired via maternal (Halsey et al., N. Engl. J. Med. 313; 544-9, 1985; Osterhaus et al., Vaccine 16: 1479-81, 1998). In this way, the vaccine virus is not provided until the passively acquired maternal antibodies have decreased to undetectable levels. In the United States, the vaccine for the measles virus is not provided until 12 to 15 months of age, once when almost all children are easily infected with the measles virus vaccine. In developing countries, the measles virus continues to have a high mortality rate, especially in children within half after the first year of life (Gellin et al., J. Infect. Dis. 170: S3-14, 1994; Taylor et al., Am. J. Epidemiol. 127: 788-94, 1988). This occurs because the measles virus, which is highly prevalent in these regions, is capable of infecting that subset of infants in whom the levels of measles-specific antibodies acquired via maternal blood have declined to a non-protective level. Therefore, there is a need for a vaccine against measles virus that is capable of inducing a protective immune response even in the presence of neutralizing antibodies of measles virus with the aim of eliminating measles virus diseases that occur within of the first year of life as well as that which is presented later. Given this need, there have been many attempts to develop an immunization strategy to protect infants in the half after the first year of life against the measles virus, although none of these strategies has been effective to date. The first strategy to develop a previous measles vaccine involved the administration of the attenuated measles virus vaccine in vivo, authorized for infants approximately six months of age by one of the following two methods (Cutts et al., Biologicals 2_5: 323-38, 1997). In a general protocol, live attenuated measles virus was administered intranasally by drops (Black et al., New Eng. J. Med. 263: 165-169; 1960; Kok et al., Trans. R. Soc. Trop.Med. Hyg. 77: 171-6, 1983; Simasathien et al., Vaccine 15: 329-34, 1997) or in the lower respiratory tract by aerosol (Sabin et al., J. Infect. Dis. 152: 1231-7, 1985), to initiate an infection of the respiratory tract. In a second protocol, the measles virus was administered parenterally, although at a higher dose than that used for the current vaccine. The administration of vaccines that can be replicated in mucosal surfaces has been successfully achieved in early infancy for vaccines of both attenuated poliovirus in vivo and rotavirus (Melnick et al., In "Fields Virology", BN Fields, DM Knipe, PM Howley, R. Chanock, JL Melnick, TP Monath, B. Roizman, and SE Straus, Eds., Vol. 1, pp. 655-712.2 vols Lippencott-Raven Publishers, Philadelphia, 1996; Perez-Schael et al., N. Engl. J. Med. 337: 1181-7, 1997), presumably because passively acquired IgG antibodies have less access to mucosal surfaces than those that do to systemic sites of viral replication. In this situation, attenuated polio vaccine viruses in vivo are capable of infecting the mucosal surface of the gastrointestinal tract or the respiratory tract of young infants, including those with maternal antibodies, resulting in the induction of a protective immune response. Therefore, a reasonable method is to immunize via the respiratory tract of the young infant with attenuated measles virus vaccine in vivo, since this is the natural route of infection with the measles virus. However, the measles virus attenuated in vivo, which is infectious by the parenteral route was inconsistently infectious by the intranasal route (Black et al., New Eng. J. Med. 2 63: 1 65- 1 69, 1 960; Cutts et al., Biologicals 25: 323-38, 1997; Kok et al., Trans. R. Soc. Trop.Med. Hyg. J7: 17 1 - 6, 1 98 3; Simasathien et al., Vaccine 1 5 : 32 9-34, 1 997), and this decreased ineffectiveness was especially evident for the Schwartz strain of the measles virus vaccine which is the current vaccine strain. Presumably,. During the attenuation of this virus by passage in the tissue culture cells of daily origin, the virus loses a significant amount of infectivity to the upper respiratory tract of humans. In effect, a purity mark of the measles virus biology is that the virus undergoes rapid changes in biological properties when it is developed in vitro. Because this relatively simple route of immunization is not successful, a second method has been tried which involves administering the live virus vaccine by aerosol in the lower respiratory tract (Cutts et al., Bioloigicals 25: 323-38, 1997; et al., J. Infect. Dis. 152: 1231-7, 1985). The infection of young infants by the aerosol administration of the measles virus vaccine was carried out in fairly controlled experimental studies, although this has not been possible to reproducibly deliver a live attenuated measles virus vaccine in the aerosol field adjustments to the young non-cooperative infant (Cutts et al., Biologicals 25: 323-38, 1997). In another attempt to immunize six-month-old infants, the measles vaccine virus was administered parenterally at an increased dose of 10 to 100 times (Markowitz et al., Engl. J. Med. 322: 580- 7, 1990). Although high titre in vivo measles vaccination improved seroconversion in infants 4 to 6 months of age, there was an increase associated with mortality in high titre vaccine recipients after infancy (Gellin et al., J Infect Dis 170: S3-14, 1994; Holt et al., Infect J. Dis. 168: 1087-96, 1993; Markowitz et al., N. Engl. J. Med. 322: 580-7 , 1990) and this approach to immunization has been abandoned.

A second strategy previously explored for a measles virus vaccine was the use of an inactivated measles virus vaccine, specifically, an inactivated formalin whose measles virus or a subunit of the virus vaccine was prepared from the measles virus ( Griffin et al., In "Fields Virology", BN Fields, DM Knipe, PM Ho law, RM C anock, JL Melnick, TP Monath, B. Roizman, and SE Straus, Eds., Vol. 1, pp. 1267- 1312. Lippincott-Raven Publishers, Philadelphia, 1996). However, the clinical use of the vaccines in the 60's revealed a very serious complication, namely that inactivated virus vaccines enhanced the disease rather than preventing it (Fulginiti et al., JAMA 202: 1075-80, 1967). ). This was first observed with the formalin inactivated measles virus vaccine (Fulginiti et al., JAMA 202: 1075-80, 1967). Initially, this vaccine prevents measles, but after several years the vaccine loses its resistance to infection. When subsequently infected with naturally circulating measles virus, the vaccines developed an atypical condition with accentuated systemic symptoms and pneumonia (Fulginiti et al., JAMA 202: 1075-80, 1967; Nader et al., J. Pediatr. 72: 22-8, 1968; Rauh et al., Am. J. Dis. Child 109: 232-7, 1965). A retrospective analysis showed that inactivation with formalin destroys the ability of the fusion protein (F) of measles to induce antibodies that inhibit hemolysis, although it does not destroy the ability of the HA protein (hemagglutinin or fixation) to induce neutralizing antibodies (Norrby et al., J. Infect. Dis. 132; 262-9, 1975; Norrby et al., Infect. Immun. 11: 231-9,1975). When the immunity induced by the HA protein has sufficiently decreased to allow extensive infection with the wild-type measles virus, an altered and sometimes more serious disease is observed at the replication sites of the measles virus (Bellanti, Pediatrics 48: 715-29, 1971; Buser, N. Engl. J. Ed. 277; 250-1, 1967). It is believed that this atypical disease is provided in part by an immune response delivered by an altered cell in which Th-2 cells are preferentially induced leading to increased manifestations of the disease at sites of viral replication (Polack et al. , Nat. Med. 5: 629-34, 1999). Due to this experience with non-living measles virus vaccines and also because the immunogenicity of these parenterally administered vaccines can be decreased by passively transferred antibodies, there has been considerable reluctance to evaluate these vaccines in human infants. It should be noted that the potentiation of the disease seems to be associated only with the exterminated vaccines. Yet another strategy that has been explored to develop a measles vaccine for use in young infants has been to use viral vectors to express a protective antigen of the measles virus (Drillien et al., Proc. Nati. Acad. Sci. USA 85: 1252-6, 1988, Fooks et al., J. Gen. Virol. 79.1027-31 1998, Schnell et al., Proc. Nati, Acad. Sci. USA 93: 11359-65, 1996a, Taylor et al., Virology. 187: 321-8, 1992; Wild et al., Vaccine: 441-2, 1990; Wild et al., J. Gen. Virol. 73: 359-67, 1992). A variety of vectors have been explored, including poxviruses such as, for example, the replication competent vaccinia virus or the modified Ankara vaccinia virus (MVA) strain, with replication defective. Competent replication vaccinia recombinants expressing the F or HA glycoprotein of measles virus were effective in immunologically natural vaccines. However, when administered parenterally in the presence of the passive antibody against the measles virus, its immunogenicity and protective efficacy was significantly canceled (Galletti et al., Vaccine 13: 197-201, 1995; Osterhaus et al., Vaccine 16 : 1479-81, 1998; Siegrist et al., Vaccine 16: 1409-14, 1998; Siegrist et al., Dev. Biol. Stand .95: 133-9, 1998).

Vaccinia recombinants with competent replication expressing the RSV protective antigens have also been shown to be ineffective in inducing a protective immune response when administered parenterally in the presence of the passive antibody (Murphy et al., J. Virol. 62_: 3907-10, 1988a), although they easily protect these hosts when they are administered intranasally. Unfortunately, competent replication vaccinia virus recombinants are not sufficiently attenuated for use in immunocompromised hosts such as, for example, persons with human immunodeficiency virus (HIV) infection (Fenner et al., World Health Organization, Geneva, 1988; Redfield et al., N. Engl. J. Med. 316: 673-676, 1987) and its administration by the intranasal route even to immunocompetent individuals could be problematic. Therefore, they are not sought as vectors for use in infants, humans, some of whom may be infected with HIV. The MVA vector, which is derived by more than 500 steps in embryonic chicken cells (Mayr et al., Infection 3: 6-14, 1975; Meyer et al., J. Gen. Virol. 7_2: 1031-1038, 1991 ), has also been evaluated as a potential vaccine vector for the protective antigens of various paramyxoviruses (Durbin et al., J. Infect. Dis. 179: 1345-51, 1999a; Wyatt et al.

Vaccine 14: 1451-1458, 1996). MVA is a fairly attenuated host classification mutant that replicates well in avian cells but not in most mammalian cells, including those obtained from monkeys and humans (Blanchard et al., J. Gen. Virol. 79: 1159-1167, 1998 / Carroll et al., Virology 238: 198-211, 1997 / Drexler et al., J. Gen. Virol. 79: 347-352, 1998 / Sutter et al., Proc. Nati. Acad. Sci. USA 89: 10847-10851, 1992). Avipox vaccine vectors, which have a host classification restriction similar to that of MVA, have also been constructed to express protective antigens against the measles virus (Taylor et al., Virology 187: 321-8, 1992). MVA is not pathogenic in immunocompromised hosts and has been administered in many humans without incident (Mayr et al., Zentralbl Bakteriol. [B] 167: 375-90, 1978 / Stickle et al., Dtsch.Med. Wochenschr. 99: 2386-92, 1974 / Werner et al., Archives of Virology 64: 247-256, 1980). Unfortunately, both the immunogenicity and the efficacy of the MVA expressing a protective antigen against paramyxoviruses were abolished in passively immunized rhesus monkeys whether or not they were delivered by a parenteral or a topical route (Durbin et al., Virology 235: 323- 332, 1999). The immunogenicity of DNA vaccines expressing parentally-provided measles virus protective antigens also decreased in passively immunized hosts (Siegrist et al., Dev. Biol. Stand 95: 133-9, 1998). Defective replication vectors expressing protective antigens against measles virus are currently being evaluated, including the recombinants HA of measles virus by adenovirus (Fooks et al., J. Gen. Virol. 79: 1027-31, 1998) . In this context, MVA recombinants expressing parainfluenza virus antigens, other than vaccinia virus recombinants with competent replication, lack protective efficacy when administered by a mucosal route to animals with passively acquired antibodies, and it is unlikely that they, or similar avipox vectors, can be used in infants with measles virus antibodies acquired via the mother. Based on the reports summarized above, it appears unlikely that a replication-defective or replication-competent poxvirus vector, or a DNA vaccine, that expresses a protective antigen for measles virus will be satisfactorily immunogenic or effective in infants who have specific antibodies to the virus. maternal measles virus passively acquired. A newly developed competent replication virus vector expressing measles virus HA that replicates in the respiratory tract of animal hosts has been developed, namely, vesicular stomatitis virus (VSV), a rhabdovirus that naturally infects cattle but not human beings (Roberts et al., J. Virol. 73.3723-32, 1999; Schnell et al., Proc. Nati Acad. Sci. USES. 93_: 11359-65, 1996a). It is known that VSV is an animal virus that can cause a disease in humans, the development of this recombinant for use in humans, will require that the VSV structure that is successfully attenuated in human infants be identified first (Roberts et al., J Viro! 73: 3723-32, 1999), but these clinical studies have not begun. Although there have been numerous advances towards the development of effective vaccine agents against PIV and other pathogens, including measles, there remains a clear need in the art for additional tools and methods to design safe and effective vaccines to alleviate the serious health problems that can be attributed to these pathogens, particularly among young infants. Among the remaining challenges in this context is the need for additional tools to generate adequately attenuated, immunogenic and genetically stable vaccine candidates for use in various clinical settings against one or more pathogens. To facilitate these objectives, existing methods to identify and incorporate attenuating mutations in recombinant vaccine strains and to develop vector-based vaccines and immunization methods should be expanded. Surprisingly, the present invention meets these needs and provides additional advantages as described hereinafter.

SUMMARY OF THE INVENTION The present invention provides chimeric parainfluenza viruses (PIVs) that are infectious in humans and other mammals and that are useful in various compositions to generate desired immune responses against one or more of the PIV , or against a PIV and one or more additional pathogens in a host susceptible to its infection. In preferred aspects, the invention provides novel methods for designing and producing chimeric, attenuated PIVs that are useful as vaccine agents for preventing and / or treating infection and related disease symptoms that can be attributed to PIV and one or more pathogens. additional Included within these aspects of the invention are the novel, isolated polynucleotide molecules and vectors incorporating these molecules comprising a chimeric PIV genome or antigenome that includes a genome or antigenome of the partial or complete PIV vector combined or integrated with one or more heterologous genes or genomic segments that code for individual or multiple antigenic determinants of a heterologous pathogen or of multiple heterologous pathogens. Also provided within the invention are methods and compositions that incorporate a chimeric PIV for the prophylaxis and treatment of infection by both selected PIV and one or more heterologous pathogens, eg, a heterologous PIV or a pathogen without PIV such as for example a measles virus. Thus, the invention includes methods and compositions for developing live vaccine candidates based on chimeras that employ a parainfluenza virus or subviral particle that is modified recombinantly to incorporate one or more antigenic determinants of a pathogen or heterologous pathogens. The chimeric PIVs of the invention are constructed through a virus recovery system based on cDNA. The recombinant chimeric PIVs produced from a cDNA replicate independently and propagate in a similar manner as biologically derived viruses. Recombinant viruses are designed to incorporate nucleotide sequences from either a PIV vector genome or antigenome (ie, a "receptor" or "background"), or from one or more heterologous "donor" sequences that code for one or more antigenic determinants of a different PIV or heterologous pathogen - to produce an infectious chimeric virus or subviral particle. In this way, the candidate vaccine viruses are recombinantly designed to produce an immune response against one or more of the PIV or a polyspecific response against a selected PIV and a pathogen without PIV in a mammalian host susceptible to infection thereof. . Preferably, the PIV pathogens and / or without PIV from which the heterologous sequences encoding the antigenic determinants are human pathogens and the host is a human host. Also, preferably, the PIV vector is a human PIV, although non-human PIV, for example a bovine PIV (BPIV), can be used as a vector to incorporate the antigenic determinants of the human and PIVs. other human pathogens. The chimeric PIVs according to the invention can produce an immune response against a specific PIV, for example, HPIV1, HPIV2, HPIV3 or a polyspecific immune response against multiple PIV, for example, HPIV1 and HPIV2. Alternatively, the chimeric PIVs of the invention can produce a polyspecific immune response against one or more of the PIV and a pathogen without PIV such as for example the measles virus. The exemplary chimeric PIV of the invention incorporates a chimeric PIV genome or antigenome as described above, as well as a nucleocapsid protein (N), a nucleocapsid phosphoprotein (P), and a large polymerase protein (L). Additional PIV proteins can be included in various combinations to provide a range of infectious subviral particles, to a complete viral particle or a viral particle containing supernumerary proteins, antigenic determinants or other additional components. The chimeric PIV of the invention includes a partial or complete "vector" PIV genome or antigen derived from or patterned after a human PIV or a non-human PIV combined with one or more heterologous genes or genomic segments from a different PIV or other pathogen to form the chimeric PIV genome or antigenome. In preferred aspects of the invention, the chimeric PIV incorporates a partial or complete human PIV vector genome or antigenome combined with one or more heterologous genes or genomic segments from a second human PIV or a pathogen without PIV such as for example the measles virus . The "vector" PIV genome or antigenome typically acts as a receptor or carrier to which one or more "donor" genes or genomic segments of a heterologous pathogen are added or incorporated. Typically, polynucleotides that encode one or more antigenic determinants of the heterologous pathogen are added to the genome or antigenome vector or substituted therein to provide a chimeric PIV which thus acquires the ability to produce an immune response in a host selected against the heterologous pathogen. In addition, the chimeric virus may exhibit other novel phenotypic characteristics as compared to one or both of the vector and heterologous PIV pathogens. For example, the addition or substitution of the heterologous genes or genomic segments within a PIV vector strain may additionally or independently, result in an increase in attenuation, changes in development or other desired phenotypic changes as compared to a phenotype. corresponding to the unmodified vector virus and / or donor. In one aspect of the invention, the chimeric PIVs are attenuated for greater efficacy as a vaccine candidate by incorporation of large polynucleotide inserts that specify the level of attenuation in the resulting chimeric virus dependent on the size of the insert. Candidates for the chimeric PIV vaccine of the invention carry one or more major antigenic determinants of a human PIV, for example, of HPIV1, HPIV2 or HPIV3 and thus produce an effective immune response against the selected PIV in human hosts. The antigenic determinant that is specific for a selected human PIV can be encoded by the genome or antigenome vector, or it can be inserted into the genome or PIV vector antigenome or bound thereto as a heterologous polynucleotide sequence of a different PIV. The main protective antigens of human PIVs are their HN and F glycoproteins, although other proteins may also contribute to a protective or therapeutic immune response. In this context, both humoral and cell-mediated immune responses are advantageously produced by candidate vaccines represented within the invention. In this way, polynucleotides encoding antigenic determinants that may be present in the genome or antigenome vector, or integrated therein as a heterologous gene or genomic segment, may encode one or more N, P, C proteins, D, V,, F, HN and / or L of the PIV or immunogenic fragments or epitopes selected from them of any human PIV. In addition to having one or more major antigenic determinants of a selected human PIV, the preferred chimeric PIV vaccine viruses of the invention carry one or more major antigenic determinants of a second human PIV or of a pathogen without PIV. In the aspects of example, the chimeric PIV includes a genome or antigenome vector which is a human or partial PIV genome (HPIV), for example HPIV3, and also includes one or more heterologous genes or Genomic segments encoding antigenic determinants of at least one heterologous PIV, for example HPIVl and / or HPIV2. Preferably, the genome or vector antigenome is a partial or complete HPIV3 genome or antigenome and the heterologous genes or genomic segments that code for the antigenic determinants are from one or more HPIV (s) heterologous. In alternative embodiments, one or more genes or genomic segments that code for one or more antigenic determinants of HPIV1 can be added to the HPIV3 genome or partial or complete antigenome or can be substituted therein. Preferably, the HPIV1 antigenic determinants are selected from HPIV1 HN and F glycoproteins or comprise one or more antigenic domains, fragments or epitopes of the HN and / or F glycoproteins. In various exemplary embodiments, both of the HPIV1 genes encoding for glycoproteins HN and F are substituted for the HN and F genes of HPIV3 counterpart in the genome or HPIV3 vector antigenome. These constructs provide the chimeric PIVs that produce a mono- or poly-specific immune response in humans for HPIV3 and / or HPIV1. In addition to the exemplary embodiments, one or more genes or genomic segments that code for one or more antigenic determinants of HPIV2 are added to or incorporated into the HPIV3 genome or antigenome, providing a new or additional immunospeci? Chimera resulting against HPIV2 alone or against HPIV3 and HPIV2. In more detailed aspects, one or more HPIV2 genes or genomic segments encoding one or more HN and / or F glycoproteins or antigenic domains, fragments or epitopes thereof are added to the partial or complete HPIV3 vector genome or antigenome or incorporated within of the same. Still in further aspects of the invention, multiple eterologous genes or genomic segments encoding antigenic determinants of multiple heterologous PIVs are added to or incorporated into the genome or antigenome PIV vector, preferably a genome or antigenome vector. HPIV. In a preferred embodiment, heterologous genes or genomic segments encoding antigenic determinants from both HPIV1 and HPIV2 are added to or incorporated into the whole or partial HPIV1 genome or antigenome vector. In more detailed aspects, one or more HPIV1 genes or genomic segments encoding one or more HN and / or F glycoproteins (or antigenic domains, fragments or epitopes thereof) and one or more HPIV2 genes or genomic segments encoding glycoproteins HN and / or F, antigenic domains, fragments or epitopes, are added to or incorporated into the genome or HPIV3 vector antigenome vector. In one example, both HPIV1 genes encoding HN and F glycoproteins are replaced by the counterpart HPIV3 HN and F genes to form a chimeric HPIV3-1 genome or antigenome vector, which is further modified by the addition or incorporation of one or more genes or genomic segments that code for individual or multiple antigenic determinants of HPIV2. This is easily accomplished within the invention, for example, by adding or substituting a transcription unit comprising an open reading frame (ORF) of a HPIV2 HN within the chimeric HPIV3-1 vector antigen or genome. Following this method, the specific constructs exemplifying the invention are provided, which produce the chimeric PIV having antigenic determinants of HPIV1 and HPIV2, as exemplified by the candidates for PIV3r-1.2HN and PIV3r-lcp45.2HN vaccine described herein later . In alternative aspects of the invention, the chimeric PIVs of the invention are based on a human PIV vector genome or antigenome that is used as a receptor for the incorporation of the major antigenic determinants from a pathogen without PIV. Pathogens from which one or more antigenic determinants may be adopted in the candidate for chimeric PIV vaccine include but are not limited to: measles virus, respiratory syncytial virus subgroup A and subgroup B, mumps virus, human papilloma virus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filovirus, bunyavirus, flavivirus , alphavirus and influenza virus. This collection of pathogens that may thus be the target for the development of vaccines according to the methods of the invention is only as an example, and those skilled in the art will understand that the use of PIV vectors to carry antigenic determinants extends widely to a larger host of additional pathogens. Thus, in various alternative aspects of the invention, a human PIV genome or antigenome can be used as a vector for the incorporation of one or more major antigenic determinants from a wide range of pathogens without PIV. Representative major antigens that can be incorporated into the chimeric PIVs of the invention include but are not limited to: HA and F proteins of measles virus; proteins F, G, SH and M2 of the respiratory syncytial virus of subgroup A and subgroup B, proteins HN and F of mumps virus, protein Ll of human papilloma virus, protein gp 160 of human immunodeficiency virus type 1 or type 2 , herpes simplex virus proteins and gB, gC, gD, gE, gG, gH, gl, gJ, gK, gL and gM of cytomegalovirus, G protein of rabies virus, gp 350 protein of Epstein Barr virus; F protein of filovirus, protein G of bunyavirus, proteins E and NS1 of flavivirus and protein E of alphavirus. Various human PIV vectors can be employed to carry heterologous antigenic determinants of pathogens without PIV to produce one or more specific or supplied humoral immune responses. cell against the antigenic determinants carried by the chimeric vaccine virus and thus produce an effective immune response against the wild type "donor" pathogen in susceptible hosts. In preferred embodiments, one or more heterologous genes or genomic segments from the donor pathogen bind to or are inserted into the partial or complete HPIV3 genome or antigenome. Alternatively, the heterologous gene or genomic segment may be incorporated into a chimeric HPIV vector genome or antigenome, for example a partial or complete HPIV3 genome or antigenome carrying one or more genes or genomic segments of a heterologous PIV. For example, genes or genomic segments that code for the antigenic determinants of a pathogen without PIV can be combined with a full or partial chimeric HPIV3-1 genome or antigenome vector or can be combined therewith, eg, as described above having one or both of the HPIV1 genes encoding HN and F glycoproteins replaced by the counterpart HPIV3 HN and F genes. Alternatively, the genes or genomic segments encoding the antigenic determinants of a pathogen without PIV can be combined with a partial or complete chimeric genome or antigenome that incorporates the individual or multiple antigenic determinants of HPIV2, eg, a Hn gene of HPIV2 within of a HPIV1 or HPIV3 vector genome or antigenome, or a chimeric HPIV3-1 vector genome or antigenome as described above. Heterologous genes or genomic segments that code for one or more antigenic determinants of measles can be combined with any of the PIV vectors or chimeric PIV vectors described herein. In the examples provided herein, the genome or antigenome vector is a partial or complete HPIV3 genome or antigenome, or a chimeric HPIV genome or antigenome that comprises a partial or complete HPIV3 genome or antigenome that has one or more genes or genomic segments that code for antigenic determinants of a heterologous HPIV added or incorporated therein. In a chimeric construct, a transcription unit comprising an open reading frame (ORF) of an HA gene of measles virus is added to a HPIV3 vector genome or antigenome in various positions, providing candidates for chimeric PIV / measles vaccine. example PlV3r (HA HN-L), PIV3r (HA NP), cp45Lr (HA NP), PIV3r (HA PM), or cp45Lr (HA PM). In further exemplary embodiments, the PIV vector genome or antigenome is a chimeric HPIV genome or antigenome comprising a partial or complete HPIV3 genome or antigenome having one or more genes or genomic segments encoding one or more antigenic determinants of HPIV1 added or incorporated in it. This construct can be used as a vector, for example, for the measles virus wherein the heterologous antigenic determinants are selected from the HA and F proteins of the measles virus and the antigenic domains, fragments and epitopes thereof. In one example, a transcription unit comprising an open reading frame (ORF) of an HA gene of measles virus is added to the HPIV3-1 genome or antigenome vector or is incorporated into it which has both of the ORFs HN and F of HPIV3 replaced by the ORFs of HN and F of HPIV1. Among this category of recombinants are candidates for vaccine identified hereinafter as PIV3r-1 HAP-M or PIV3r-1 HAP-M cp45L. In other detailed embodiments of the invention, the complete partial PIV vector genome or antigenome is combined with one or more heterologous genes or "supernumerary" genomic segments (ie, in addition to a total gene complement, if present in a wild-type or mutant vector, for example). example, a chimeric vector structure) to form the chimeric PIV genome or antigenome. The genome or vector antigenome is often a complete HPIV3 or HPIV3-1 chimeric genome or antigenome and supernumerary heterologous genomic genes or segments are selected from HPIV1 HN, HPIV1 F, HPIV2 HN, HPIV2 F, measles HA and / or a translationally silent synthetic gene unit. In certain exemplary embodiments, one or both of the HPIV1 HN ORF and / or HPV2 HN ORF are inserted into the HPIV3 vector antigenome or genome, respectively. In more detailed embodiments, the HPIV1 HN ORFs, HPIV2 HNs and measles virus HAs are inserted between the N / P, P / M and HN / L genes respectively. Alternatively, HPIV1 HN and HPV2 HN genes can be inserted between the N / P and P / M genes, respectively and a 3918-nt GU insert is added between the HN and L genes. Among this category of recombinants are the candidates for vaccine identified hereinafter as HPIV3r 1HNN-P, HPIV3r 1HNP-M, HPIV3r 2HNN-P, HPIV3r 2HNP-M, HPIV3r 1HNN-P2HNP-M, HPIV3r 1HNN-P2HNP-M HAHN-L and HPIV3r 1HNN -P 2HNP-M 3918GUHN-L. In this way designed and constructed, the chimeric PIV of the invention can contain protective antigens of one, two, three, four or more different pathogens. For example, candidates are provided for vaccine containing protective antigens from one to four pathogens selected from HPIV3, HPIV1, HPIV2, and measles virus * To construct these candidates for multi-specific vaccine, one or more supernumerary heterologous genes can be added or genomic segments to which a total supernumerary foreign sequence length can be added to the recombinant genome or antigenome from 30% to 50% or greater (for example in comparison to the length of the wild type HPIV3 genome of 15,462 nt). The addition of one or more supernumerary heterologous genes or genomic segments in this context often specifies a chimeric PIV attenuation phenotype, which exhibits at least a 10- to 100-fold decrease in replication, often from 100 to 1,000-fold and up to 1,000 to 10,000 times or more in the upper and / or lower respiratory tract. To chimeric PIV clones of construction, a heterologous gene or genomic segment of a donor PIV or pathogens without PIV can be added or substituted at any position operable in the genome or antigenome vector. Frequently, the position of a gene or the replacement of the genomic segment will correspond to a position of the order of the wild-type gene of a gene or genome segment within the genome or partial or complete PIV vector antigenome. In other embodiments, the heterologous gene or genomic segment is added or substituted in a position that is more promotora -primary or promoter -relative as compared to an order position of the wild-type gene of a counterpart gene or genomic segment within the background genome or antigenome, to intensify or reduce the expression, respectively, of the heterologous gene or genomic segment. In more detailed aspects of the invention, a heterologous genomic segment, for example a genomic segment encoding an immunogenic ectodomain of a heterologous or pathogen PIV without PIV, can be replaced by a corresponding genomic segment in a counterpart gene in the genome or antigenome PIV vector to provide constructs coding for chimeric proteins, for example, fusion proteins having a cytoplasmic tail and / or a transmembrane domain of a PIV fused to an ectodomain of another PIV or pathogen without PIV. In alternate embodiments, a chimeric PIV genome or antigenome can be designed to encode a polyspecific chimeric glycoprotein in the recombinant virus or subviral particle having inmunglenic glycoprotein domains or epitopes from two different pathogens. Still in additional modalities, heterologous genes or genomic segments from a PIV or pathogens without PIV (ie without substitution) can be added within a genome or PIV vector antigenome to create novel immunogenic properties within the resulting cloning. In these cases, the heterologous gene or genomic segment may be added as a gene or supernumerary genomic segment, optionally for the additional purpose of attenuating the resulting chimeric virus, in combination with a genome or PIV vector antigenome. Alternatively, the heterologous gene or genomic segment can be added together with the deletion of a selected gene or genomic segment in the genome or antigenome vector. In preferred embodiments of the invention, the heterologous gene or genomic segment is added in an intergenic position within a genome or partial or complete PIV vector antigenome. Alternatively, the gene or genomic segment can be inserted into other non-coding regions of the genome, for example, within 5 'or 3' non-coding regions or at other positions where non-coding nucleotides occur within the genome or antigenome vector. In some cases, it may be desired to insert the heterologous gene or genomic segment into a non-coding site corresponding to a cis-acting or overlapping regulatory sequence within the genome or antigenome vector, for example, within a sequence required for a efficient replication, transcription and / or translation. These regions of the genome or antigenome vector represent target sites for the disruption or modification of regulatory functions associated with the introduction of the heterologous gene or genomic segment. For the preferred purpose of the candidate vaccine virus construct - for clinical use, it is often desirable to adjust the chimeric PIV attenuation phenotype of the invention by introducing additional mutations that increase or decrease the level of attenuation in the recombinant virus. Therefore, in further aspects of the invention, attenuated chimeric PIVs are produced in which the chimeric genome or antigenome is further modified by introducing one or more attenuating mutations that specify an attenuation phenotype in the resulting virus or subviral particle. These attenuating mutations can be generated de novo and tested for attenuating effects according to well-known rational mutagenesis design strategies. Alternatively / attenuating mutations can be identified in biologically existing derived mutant PIV or other viruses and thereafter incorporated into a chimeric PIV of the invention. Preferred attenuating mutations in the above context are easily identified and incorporated into a chimeric PIV, either by inserting the mutation into the. g.enoma or vector antigenome when cloning or mutagenizing the vector genome or antigenome to contain the attenuating mutation. Preferably, attenuating mutations are designed within the genome or antigenome vector and se. import or copy from attenuated PIV mutants, biologically derived. These are recognized to include, for example: PIV mutants with cold passage (cp), adapted to cold (ca), restricted to the host classification (hr),. plate small (sp) and / or sensitive to temperature (ts). In exemplary embodiments, one or more attenuating mutations present in the well-characterized JS HPIV3 cp45 mutant strain are incorporated within the chimeric PIV of the invention, preferably including one or more mutations identified in the L-polymerase protein, for example, in a corresponding position yrg42 Leug92 or Thrissa of JS. Alternatively, attenuating mutations present in the cp45 mutant strain of JS HPIV3 are introduced into the N protein of the chimeric PIV clones, for example encoding amino acid substitutions at a position corresponding to the Valg6 or Ser3B9 residues of JS. Still additional useful mutations encode for amino acid substitutions in. the protein. C, for example, in a position corresponding to? 1ß9β of JS and in the protein, for example, in a position corresponding to P oigg (for example a Proigg for the Thr mutation). Other mutations identified in cp45 PIV3 JS that can be adopted to adjust the attenuation of a chimeric PIV of the invention are found in the F protein, for example, in a position corresponding to 1.1e 4.20 or Ala45o of JS and in the HN protein , for example, in a position corresponding to the residue Val384 of JS. Attenuating mutations of biologically derived PIV mutants for chimeric PIV incorporation of the invention also include mutations in non-coding portions of the PIV genome or antigenome, for example, in a 3 'leader sequence. Exemplary mutations in this context can be designed at a position on the 3 'leader of a recombinant virus at a position corresponding to nucleotide 23, 24, 28 or 45 of JS cp45. Still further example mutations can be designed in the start sequence of the N gene, for example, by changing one or more nucleotides in the start sequence of the N gene, for example, in a position corresponding to nucleotide 62 of JS cp45. From PIV3 JS cp45 and other mutants ^ PIV biologically derived, a large "menu" of attenuating mutations is provided, each of which can be combined with any other mutations to finely adjust the level of attenuation in the candidates for chimeric PIV vaccine of the invention. In modalities of 10 example, chimeric PIVs are constructed They include one or more and preferably two or more mutations of HPIV3 JS cp45. In this way, the chimeric PIVs of the invention. selected for use with the vaccine often have two and Sometimes three or more attenuating mutations from biologically derived PIV mutants or similar model sources, to achieve a satisfactory level of attenuation of broad clinical use.

^ Preferably, these attenuating mutations 20 incorporated within the recombinant chimeric PIVs of the invention are stabilized by multiple nucleotide substitutions at a codon specifying the mutation. The introduction of mitigating mutations and Other desired phenotype specifiers in a selected PIV vector, including chimeric bovine-human PIV vectors, can be achieved by transferring a heterologous gene or genomic segment containing the mutation, eg, a gene encoding a mutant L protein, or portion thereof, in the genome or PIV vector antigenome. Alternatively, the mutation may be present in the selected genome or antigenome vector and the heterologous gene or introduced genomic segment may not carry mutations or may carry one or more additional different mutations. In certain examples, the genome or antigenome vector is modified at one or more sites corresponding to a mutation site in a heterologous "donor" virus (e.g., a negative-strand RNA virus with PIV or without PIV, bovine or human, heterologous ) to contain or encode the same, or a conservatively related mutation (eg, a conservative amino acid substitution) as a mutation identified in the donor virus (see, PCT / USOO / 09695 filed on April 12, 2000 and its Priority of U.S. Provisional Patent Application Serial No. 60 / 129,006, filed April 13, 1999, incorporated herein by reference). In an exemplary embodiment, a PIV vector genome or antigenome is modified at one or more sites corresponding to the mutation site in HPIV3 JS cp45, as mentioned above, to contain or encode the same or a conservatively related mutation as identified in the "donor" cp45.

Preferred mutant PIV strains for identifying and incorporating attenuating mutations in the PIV vectors of the invention include cold-pass (cp), cold-adapted (ca) mutants, restricted to host classification (hr), small plate (sp) and / or temperature sensitive (ts), for example strain of mutant JS HPIV3 cp45. Attenuating mutations from the biologically derived PIV mutants for incorporation into chimeric human-bovine PIV of the invention also include mutations in the non-coding portions of the PIV genome or antigenome, for example in a 3 'leader sequence. Exemplary mutations in this context can be designed at a position in the 3 'guiding sequence of a recombinant virus at a position corresponding to nucleotide 23, 24, 28 or 45 of JS cp45. Still further exemplary mutations can be designed in the start sequence of the N gene, for example by changing one or more nucleotides in the start sequence of the N gene, for example, in a position corresponding to nucleotide 62 of JS cp45. Additional mutations that can be adopted or transferred to the PIV vectors of the invention can be identified in unsegmented negative strand RNA viruses without PIV and incorporated into the PIV mutants of the invention. This can be easily accomplished by mapping the mutation identified in a heterologous negative-strand RNA virus to a corresponding homologous site in a recipient PIV genome or antigenome and mutating the sequence existing in the receptor for the mutant genotype (either by a identical or conservative mutation), as described in PCT / USOO / 09695 filed on April 12, 2000, and its United States Provisional Patent Application Priority Serial No. 60 / 129,006, filed April 13, 1999, incorporated herein by reference reference. In accordance with this disclosure, additional attenuating mutations can be easily adopted or designed within the chimeric PIVs of the invention that are identified in other viruses, in particular other unsegmented negative chain RNA viruses. Still in further aspects of the invention, the chimeric PIVs, with or without attenuating mutations modeled after the biologically derived attenuated mutant viruses, are constructed to have additional nucleotide modifications to provide a structural, or functional, phenotypic, desired change. Typically, the modification of the selected nucleotide will be done within the partial or complete PIV vector genome, although these modifications can also be made within any heterologous gene or genomic segment that contributes to chimeric cloning. Preferably, these modifications specify a desired phenotypic change, for example a change in development characteristics, attenuation, temperature sensitivity, cold adaptation, plate size, host classification restriction or immunogenicity. Structural changes in this context include the introduction or removal of restriction sites in PIV coding for cDNAs for ease of handling and identification. In preferred embodiments, nucleotide changes within the genome or antigenome of a chimeric PIV include modification of a viral gene by partial or complete deletion of the gene or reduction or removal (separation) of its expression. The target genes for the mutation in this context include any of the PIV genes, which include the N nucleocapsid protein, the phosphoprotein P, the large polymerase L subunit, the M protein matrix, the HN protein hemagglutinin-neuramidase, the F protein fusion and the products of open reading frames C, D and V (ORF). To the extent that the recompiling virus remains viable and infectious, each of these proteins can be selectively suppressed, replaced or rearranged, in whole or in part, alone or in combination with other desired modifications, to achieve the suppression or separation of the mutants. For example, one or more of the C, D and / or V genes can be deleted in whole or in part, or their expression can be reduced or removed (for example by introducing a stop codon, by a mutation in the RNA editing site, by a mutation that alters the amino acid specified by an initiation codon or by a frame shift mutation in the target ORF (s)). In one embodiment, a mutation can be made at the editing site that prevents editing and separates the expression of the proteins whose mRNA is generated by RNA editing (Kato et al., EMBO 16: 578-587, 1997 and Schneider et al., Virology 227: 314-322, 1997, incorporated herein by reference). Alternatively, one or more of the C, D and / or V ORF (s) may be deleted in whole or in part to alter the phenotype of the resulting recombinant cloning to enhance the development, attenuation, immunogenicity or other desired phenotypic characteristics ( see U.S. Patent Application Serial No. 09 / 350,821, filed by Durbin et al on July 9, 1999, incorporated herein by reference). Alternative nucleotide modifications in the chimeric PIV of the invention include a deletion, insertion, addition or rearrangement of a cis-acting regulatory sequence for a selected gene in the recombinant genome or antigenome. In one example, a cis-acting regulatory sequence of a PIV gene is changed to correspond to a heterologous regulatory sequence, which may be a cis-acting counterpart regulatory sequence of the same gene in a different PIV, or a cis-acting regulatory sequence of a different PIV gene. For example, an extreme signal of the gene can be modified by conversion or substitution to an extreme signal of the gene of a different gene in the same PIV strain. In other embodiments, the nucleotide modification may comprise an insertion, deletion, substitution or rearrangement of a translational start site within the recombinant genome or antigenome, for example, to separate an alternative translational start site for a selected form of a protein. In addition, a variety of other genetic alterations can be produced in a chimeric PIV genome or antigenome, alone or together with one or more attenuating mutations adopted from a biologically derived PIV mutant. For example, genes or genomic segments from sources without PIV can be inserted in whole or in part. In one aspect, the invention provides methods for attenuating candidates for chimeric PIV vaccine based on host classification effects due to the introduction of one or more genes or genomic segments originating, for example, from a non-human PIV in a chimeric virus based on in a human PIV vector. For example, host classification attenuation can be conferred on a chimeric construct based on an HPIV vector by introducing nucleotide sequences from a bovine PIV (BPIV) (see, for example, as described in the Application by the United States). United Serial No. 09 / 586,479, filed June 1, 2000, which corresponds to United States Provisional Application Serial No. 60/143, 134 filed July 9, 1999, incorporated herein by reference ). These effects are attributed to the structural and functional divergence between the vector and the donor viruses and provide a stable basis for attenuation. For example, between HPIV3 and BPIV3 the percentage of amino acids identified for each of the N proteins is 86%, for P it is 65%, M 93%, F 83%, HN 77% and L 91%. All of these proteins are therefore candidates for introduction into a BPIV vector to provide an attenuated chimeric virus that is not already altered by reversion. In exemplary embodiments, the genome or antigenome vector is an HPIV3 genome or antigen and the heterologous gene or genomic segment is an ORF N derived from a BPIV3 strain. In this way, chimeric PIVs are provided within the invention based on a genome or antigenome vector which is a chimeric human-bovine PIV genome or antigenome. In certain embodiments, the chimeric human-bovine vector genome or antigenome is combined with one or more heterologous genes or genomic segments that code for one or more antigenic determinants of a heterologous pathogen selected from measles virus, the respiratory syncytial viruses of subgroup A and subgroup B, mumps virus, human papilloma virus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filovirus, bunyavirus, flavivirus, alphavirus and influenza viruses. In alternative aspects of the invention, a chimeric human-bovine vector genome or antigenome comprises a partial or complete HPIV genome or antigenome combined with one or more heterologous genes or genomic segments of a BPIV. In an exemplary embodiment, a transcription unit comprising an open reading frame (ORF) of an ORF N of BPIV3 is replaced in the genome or vector antigenome for a corresponding ORF N of an HPIV3 vector genome. Using this and similar constructs, the genome or antigenome vector is combined with an HA gene of the measles virus, or an antigenic determinant selected from another pathogen, as a supernumerary gene insert, as exemplified by the vaccine candidate identified below as HPIV3r-NB HAP-M. In other alternative aspects of the invention, the human-bovine chimeric vector genome or antigenome comprises a partial or complete HPIV genome or antigenome combined with one or more heterologous genes or genomic segments of a BPIV. For example, one or more HPIV genes or genomic segments encoding HN and / or F glycoproteins or one or more immunogenic domains, fragments or epitopes can be added to the partial or complete bovine genome or antigenome or can be incorporated therein to form the genome or antigenome vector. In certain embodiments, both HPIV3 genes encoding the HN and F glycoproteins are substituted for the HN and F genes of BPIV3 to form the genome or antigenome vector. Using this and similar constructs, the genome or antigenome vector is combined with a F and / or G gene of RSV, or an antigenic determinant selected from another pathogen, as a supernumerary gene insert, as exemplified by the vaccine candidates identified above. forward as BHPIV3r-Gl or B / HPIV3r-Fl. Still in more detailed embodiments, a chimeric human-bovine vector incorporates one or more HN and / or F genes of HPIV1 or genomic segments that code for one or more immunogenic domains, fragments or epitopes thereof and the vector is further modified by incorporating one or more HN and / or F genes of HPIV2 or genomic segments that code for one or more immunogenic domains, fragments or epitopes thereof to form the chimeric genome or antigenome that expresses protective antigens from both HPVI and HPIV2. This category of chimeric PIV is exemplified by various vaccine candidates identified below as B / HPIV3.1r-2F; B / HPIV3. lr-2HN; or B / HPIV3.1r-2F, 2HN. In still further aspects of the invention, the order of the genes can be changed to cause attenuation or reduce or intensify the expression of a particular gene. Alternatively, a PIV genome promoter can be replaced with its antigenomic counterpart to provide additional desired phenotypic changes. Different or additional modifications can be made to the recombinant genome or antigenome to facilitate manipulations, such as, for example, the insertion of single restriction sites in various intergenic regions or elsewhere. The untranslated gene sequences can be deleted to increase the insertion capacity of the foreign sequences. In still further aspects, the polynucleotide molecules or vectors encoding the chimeric PIV genome or antigenome can be modified to encode sequences without PIV, eg, a cytosine, an auxiliary epitope T, a restriction site masker or a protein or immunogenic epitope of a microbial pathogen (e.g., virus, bacterium or fungus) capable of producing a protective immune response in a targeted host. In one embodiment, chimeric PIVs are constructed to incorporate a gene that codes for a cytosine to provide novel phenotypic and immunogenic effects in the resulting chimera. In addition to providing chimeric PIV for vaccine use, the invention provides related cDNA clones and vectors that incorporate a PIV vector genome or antigenome and the heterologous polynucleotides that encode one or more heterologous antigenic determinants, wherein the clones and vectors incorporate optionally mutations and related modifications that specify one or more attenuating mutations or other phenotypic changes as described above. Heterologous sequences encoding antigenic determinants and / or specifying the desired phenotypic changes are introduced into selected combinations, for example, into an isolated polynucleotide that is a genome or recombinant cDNA antigenome vector, to produce a subviral, infectious virus or particle. , duly attenuated according to the methods described herein. These methods, coupled with routine phenotypic evaluation, provide a large collection of chimeric PIVs that have these desired characteristics such as attenuation, temperature sensitivity, altered immunogenicity, cold adaptation, small plate size, host classification restriction, genetic stability , etc. Preferred vaccine viruses among these candidates are attenuated and even sufficiently immunogenic to produce a protective immune response in the vaccinated mammal host. In related aspects of the invention, compositions (e.g., isolated polynucleotides and vectors that incorporate a cDNA encoding chimeric PIV) and methods are provided to produce an isolated infectious chimeric PIV, are included within these aspects of the invention. invention novel novel polynucleotide molecules and vectors incorporating these molecules comprising a chimeric PIV antigen or genome. Also provided is the same or different expression vector comprising one or more isolated polynucleotide molecules, encoding N, P and L proteins. These proteins may alternatively be expressed directly from the genome or antigenic cDNA. The vectors are preferably expressed or coexpressed in a cell lysate or cell free, thereby producing a subviral particle or particle of the infectious chimeric parainfluenza virus. The above methods and compositions for producing chimeric PIV provide infectious viral or subviral particles, or derivatives thereof. An infectious virus is comparable to the authentic PIV particle and is infectious like this. It can directly infect fresh cells. An infectious subviral particle is typically a subcomponent of the virus particle that can initiate an infection under appropriate conditions. For example, a nucleocapsid containing the genomic or antigenomic ARN and the N, P and L proteins in an example of a subviral particle that can initiate an infection if it is introduced into the cytoplasm of the cells. The subviral particles provided within the invention include viral particles that lack one or more proteins, protein segments or other viral components not essential to carry out the infection. In other embodiments, the invention provides a cell-free or cell lysate containing an expression vector comprising an isolated polynucleotide molecule comprising a chimeric PIV genome or antigenome as described above and an expression vector (the same vector or one different) comprising one or more isolated polynucleotide molecules encoding the NIV, P and L proteins of PIV. One or more of these proteins can also be expressed from the genome cDNA or antigenome. At the time of expression, the genome or antigenome and the N, P and L proteins combine to produce a virus or subviral particle of the infectious chimeric parainfluenza virus. In other embodiments of the invention, there is provided a cell-free or cell-free expression system (eg, a cell-free Used) that incorporates an expression vector comprising an isolated polynucleotide molecule that encodes a chimeric PIV and a vector of expression comprising one or more isolated polynucleotide molecules encoding the N, P and L proteins of a PIV. At the time of expression, the genome or antigenome and the N, P and L proteins combine to produce an infectious PIV particle, such as for example a viral or subviral particle. The chimeric PIVs of the invention are useful in various compositions to generate a desired immune response against one or more of the PIV, or against PIV and a pathogen without PIV, in a host susceptible to infection thereof. The chimeric PIV recombinants are capable of producing a mono- or polyspecific protective immune response in an infected mammalian host, and are even sufficiently attenuated so as not to cause unacceptable symptoms of the disease in the immunized host. The attenuated subviral virus or particle may be present in a cell culture supernatant, isolated from the culture or partially or completely purified. The virus can be lyophilized and can be combined with a variety of other components to be stored or delivered to a host as desired. The invention further provides novel vaccines comprising a physiologically acceptable carrier and / or an adjuvant and an isolated, attenuated, chimeric parainfluenza subviral virus or particle, as described above. In preferred embodiments, the vaccine comprises a chimeric PIV having at least one, and preferably two or more additional mutations or other nucleotide modifications that specify a balance of adequate attenuation and immunogenicity. The vaccine can be formulated in a dose of 103 to 107 PFU or attenuated virus. The vaccine may comprise attenuated chimeric PIV that produces an immune response against a single PIV strain or against multiple PIV strains or groups. In this respect, the chimeric PIV can be combined in the vaccine formulations with other PIV vaccine strains or with other viruses for viral vaccine such as for example RSV.

In related aspects, the invention provides a method for stimulating the immune system of an individual to produce an immune response against one or more of the PIV, or against a PIV and a pathogen without PIV, in a mammalian subject. The method comprises administering a formulation of an immunologically sufficient amount of a chimeric PIV in a physiologically accele carrier and / or adjuvant. In one embodiment, the immunogenic composition is a vaccine comprising a chimeric PIV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications that specify a desired phenotype and / or attenuation level as described above. The vaccine can be formulated in a dose of 103 to 107 PFU of attenuated virus. The vaccine may comprise an attenuated chimeric PIV that elicits an immune response against a single PIV, against multiple PIVs, for example, HPIV1 and HPIV3, or against one or more PIV (s) and a pathogen without PIV such as for example measles or RSV. In this context, chimeric PIVs can produce a monospecific immune response or a polyspecific immune response against multiple PIVs or against one or more of the PIVs and a pathogen without PIV. Alternatively, the chimeric PIV having different immunogenic characteristics can be combined in a vaccine mixture or administered separately in a coordinated treatment protocol to produce more effective protection against a PIV, against multiple PIV, or against one or more of the PIV and a pathogen without PIV such as for example measles or RSV. Preferably, the immunogenic compositions of the invention are administered to the upper respiratory tract, for example, by spray, drops or aerosol. Preferably, the immunogenic composition is administered to the upper respiratory tract, for example, by spray, drops or aerosol. The invention also provides novel combinatorial vaccines and coordinated vaccination protocols for multiple pathogenic agents, including multiple PIV and / or PIV and a pathogen without PIV. For example, targets selected for early vaccination according to these compositions include RSV and PIV3, which each cause a significant number of conditions within the first four months of life, while the majority of conditions caused by PIV1 and PIV2 occur after six months of age (Collins et al., In Fields Virology, Vol. 1, pp. 1205-1243, Lippincott-Raven Publishers, Philadelphia, 1996; Reed et al., J. Infect. Dis. 175: 807-13, 1997). A preferred immunization sequence employing live attenuated RSV and PIV vaccines is to administer RSV and PIV3 as early as one month of age (eg, at one and two months of age) followed by a bivalent vaccine PIV1 and PIV2 at four and six months of age. In this way it is desirable to employ the methods of the invention to administer multiple PIV vaccines, including one or more chimeric PIV vaccines, in a coordinated manner, for example, simultaneously in a mixture or separately in a defined time sequence (eg example, in a daily or weekly sequence), wherein each vaccine virus preferably expresses a different heterologous protective antigen. This coordinated / sequential immunization strategy, which is capable of inducing secondary antibody responses to multiple viral respiratory pathogens, provides a fairly powerful and extremely flexible immunization regime that is driven by the need to immunize against each of the three PIV viruses and other pathogens in early childhood. Importantly, the presence of multiple PIV serotypes and their unique epidemiology with PIV3 disease that occurs at a younger age than that of PIV1 and PIV2 makes it desirable to sequentially immunize an infant with different PIV vectors, each expressing the same heterologous antigenic determinant such as for example, the HA of the measles virus. This sequential immunization allows for the induction of the high titre of the antibody to the heterologous protein that is characteristic of the response to the secondary antibody. In one embodiment, young infants (eg, 2 to 4 month old infants) are immunized with an attenuated chimeric virus of the invention, for example a chimeric HPIV3 that expresses the measles virus HA protein and is also adapted to produce an immune response against HPIV3, such as for example rcp45L. { HA P-). Subsequently, for example at four months of age, the infant is immunized again but with a secondary, different vector construct, such as, for example, the PIV3r-1 cp45L virus expressing the HA gene of the measles virus and the antigenic determinants HPIV1. as the essential, functional glycoproteins of the vector. After the first vaccination, the vaccine will produce a primary antibody response to both the HN and F proteins of PIV3 and to the HA protein of measles virus, but not to the HN and F protein of PIV1. At the time of secondary immunization with PIV3r-l cp45L that expresses the measles virus HA, the vaccine will already be infected with vaccinia due to the absence of the antibody to the PIV1 HN and F proteins and will develop both an antibody response primary the protective HN and F antigens of PIV1 as a high secondary antibody response titrated to the HA protein of the heterologous measles virus. A similar sequential immunization scheme can be developed wherein the immunity occurs sequentially against HPIV3 and then HPIV2 by one or more of the chimeric vaccine viruses described herein, simultaneously with the stimulation of a high, primary protective titrated response and then secondary to measles or another pathogen without PIV. This sequential immunization strategy, preferably employing different serotypes of PIV as primary and secondary vectors, effectively avoids the immunity that is induced for the primary vector, a factor that ultimately limits the usefulness of vectors with only one serotype. The success of sequential immunization with the vaccine candidates of the PIV3r and PIV3r-1 viruses has been demonstrated as described above (Tao et al., Vaccine 12: 1100-8, 1999).

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB illustrate the insertion of the HA gene of measles in the HPIV3 genome (Note: all the figures presented herein and the related descriptions refer to the antigenome in the positive sense of HPIV3, 5 'to 3 ') · Figure 1A provides a diagram (top, unscaled) of insert 1926 nt containing the complete open reading frame of the Edmonston wild type hemagglutinin (HA) gene from measles virus designed to express the HA of the measles from an extra transcriptional unit. The insert contains, in order 5 'to 3': a site A // II; nts 3699-3731 of the HPIV3 antigenome containing the P / M gene binding, which includes the non-coding sequence downstream of the P gene, its end-of-gene signal, the intergenic region, and the gene start signal; three additional nts (GCG); ORF HA of the complete measles virus; HPIV3 nt 3594-3623 of the non-coding region downstream of the P gene; and a second site A // II. Panel 1 of Figure 1A illustrates the complete antigenome of the wild-type JS strain of HPIV3 (PIV3r) with the A // II site introduced into the 3 'non-coding region of the N (higher) and then (lower) N gene. the insertion of the ORF HA of measles. Panel 2 of Figure 1A illustrates the complete antigenome of the wild-type JS strain of HPIV3 (PIV3r) with the A // II site introduced into the 3 'non-coding region of the P (higher) and then (lower) P gene. the insertion of the ORF HA of measles. . SEQ ID NO. 1 and SEQ ID NO. 2 in Figure 1A. Figure IB provides a diagram (top, unscaled) of insert 2028 nt containing the complete ORF of the HA gene of measles virus. The insert contains, in order 5 'to 3': a Stul site; nts 8602 to 8620 of the HPIV3 antigenome consisting of the non-coding sequence downstream of the HN gene and its end signal of the gene; the preserved HPIV3 intergenic trinucleotide; nts 6733 to 6805 of the HPIV3 antigenome containing the start of the HN gene and the non-coding region in the 5 'direction; the ORF HA of the measles virus; HPIV3 nts 8525-8597, which are non-coding sequences downstream of the HN gene; and a second Stul site. The construct is designed to regenerate, at the time of insertion, the HN gene of HPIV3 containing the Stul site and the ORF site of the measles virus directly flanked by the transcription signals and the non-coding region of the HN gene of HPIV3. . The complete wild type HPIV3 JS antigenome (PIV3r) with the Stul site introduced at position nt 8600 in the 3 'non-coding region of the HN gene is illustrated in the following diagram (in half); in the lower part is the HPIV3 antigenome that expresses the measles HA protein inserted in the Stul site. The HA cDNA used for this insertion comes from an existing plasmid, instead of the Edmonston wild type measles virus, which was used for the insertions in the N / P and P / M regions. This cDNA had two amino acid differences from the HA protein inserted in Figure 1A, and its location in the HA gene of measles virus is indicated by the asterisks in Figure IB. SEQ ID NO. 3 and 4 are shown in Figure IB. Figure 2 illustrates the expression of the HA protein of measles virus by means of chimeric HA viruses of measles virus HPIV3r in LLC-MK2 cells. The figure represents a radioimmunoprecipitation (RIPA) analysis demonstrating that the measles HA protein is expressed by the recombinant chimeric viruses cp45Lr (HA PM) and cp45Lr (HA NP), and by the wild type strain Edmonston of measles virus (Measles), although not by wild-type HPIV3 of JSr (JSr). Infected cell lysates, labeled by the A-35S lines were inumonoprecipitated by a mixture of the three monoclonal antibodies specific for the HPIV3 HN protein. The 64kD band corresponding to the HN protein (empty arrow) is present in each of the three cell lysates infected with HPIV3 (Lines 3, 5 and 7), although not in the cell lysates infected by the measles virus (Line 9), which confirm that the chimeras cp45Lr (HA P-M) and cp45Lr (HA N-P) are really HPIV3 and express similar levels of HN proteins. Infected cell lysates labeled with Lines (b) -35S were immunoprecipitated by a monoclonal antibody mixture that recognizes the measles virus HA glycoprotein (79-XV-V17, 80-III-B2, 81-1- 366) (Hummel et al., J. Virol. 69: 1913-6, 1995; Sheshberadaran et al., Arch. Vlrol. 83: 251-68, 1985, each is incorporated herein by reference). The 76kD band corresponding to the HA protein (filled arrow) is present in the lysates from cells infected with the chimeric viruses cp45Lr (HA) (Lines 6, 8) and the measles virus (Line 10), although not in the lysates from cells infected with JSr (Line 4), a wild type HPIV3 virus that does not code for an HA gene of measles virus. Figure 3 illustrates the insertion of the HN gene of HPIV2 as an extra + transcription / translation unit in the antigenomic cDNA encoding the chimeric cp45 virus of PIV3r-lo PIV3r-1 (Note: PIV3r-1 is a PIV3r in the which genes HN and F are replaced by those cp45 of HPIV1 and PIV3r-l is a version that contains, in addition, 12 mutations from attenuated viruses cp45). The HN gene of HPIV2 was amplified from HPIV2 RNAv using RT-PCR with HPV2 specific HN gene primers (Panel TO) . The amplified cDNA, which carries a Ncol site introduced into the primer and its 5 'end and a HindIII site at its 3' end, was digested with NcoI-HindIII and ligated into pLit. PIV3 lHNhc, which would have been digested with NcoI-HindIII, to generate pLit. IV32HNhc (Panel B). The plasmid pLit. PIV32HNhc was used as a template to produce a modified PIV2 HN cassette (Panel C), which had a site at its 5 'PpuMI end and a PpuMI site introduced at its 3' end. This cassette contained, from right to left: the PpuMI site at the 5 'end, a partial 5' untranslated region (UTR) of HN of PIV3, the HN ORF of PIV2, a 3 'UTR of HN of PIV3, the sequence of the end of the gene, intergenic, of the start of the gene that exists in the binding of the HN and L gene of PIV3, a portion of the 5'-untranslated region of L of PIV3, and the PpuMI site introduced at the 3 'end. This cDNA cassette was digested with PpuMI and then ligated to p38 'APIV31hc. that had been digested with PpuMI, to generate p38 · ???? 31] ??. 2 ?? (Panel D). The 8.5Kb SspEI-Sphl fragment was assembled in the BspEI-Spnl window of pFLC.2G + .hc or pFLCcp45 to generate the final full length antigenomic cDNA, pFLC.3-lhc.2HN (Panel E) or pFLC.3-lhc .cp45.2HN (Panel F), respectively. pFLC.2G + .hc and pFLCcp45 are full-length antigenic clones encoding wild-type PIV3r-ly and PIV3rcp45, respectively, which have already been described previously (Skiadopoulos et al., J. Virol. 73: 1374-81, 1999a; Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference). Figure 4 details and verifies the construction of the PIV3r-1.2HN chimeric virus carrying the ORF HN insert of PIV2 between the F and HN genes of PIV1. Panel A represents the differences in the structures of PIV3r-l and PIV3r-1.2HN, which contains the ORF HN insert of PIV2 between the ORF F and HN of PIV1 of PIV3r-l. The arrows indicate the approximate locations of the RT-PCR primers used to amplify the fragments analyzed in panels B-D. Panels B and C represent the expected sizes of the restriction enzyme digestion fragments generated from the amplified RT-PCR products of PIV3r-ly and PIV3r-1.2HN using either the restriction endonucleases PpuMI or Ncol, with the sizes of the fragment indicated in base pairs (bp), and the results presented in Panel D. The vRNA extracted from the virus collected from PIV3r-1.2HN or LLC-K2 cells infected with PIV3r-l, was used as a template in presence and nce of reverse transcriptase (RT), to amplify the cDNA fragments by PCR using the primers indicated in Panel A. PCR fragments were nt in the RT-PCR reactions that lack RT what indicates that the template used for the amplification of the DNA fragments was RNA and did not contaminate the cDNA (Lines A and C of Panel D). When the RT step was included, PIV3r-1.2HN RNAv (Line B) provided a fragment that was approximately 2kb greater than that of its precursor PIV3r-l (Line D) indicating the presence of a 2kb insert. In addition, digestion of this 3kb fragment with several different restriction endonucleases indicates that the RT-PCR fragment from PIV3r-1.2HN (Lines with odd numbers) has patterns that are different from those of the PIV3r-l precursor (Lines with even numbers ) for each restriction endonuclease tested. For each digestion, the number of sites and the sizes of the obtained fragments were consistent with the predicted sequence of the RT-PCR products of PIV3r-l and PIV3r-1.2HN representative examples are presented. First, the PpuMI digestion of the RT-PCR product from PIV3r-1.2HN (Line 1) yielded three bands of the expected sizes that indicate the presence of two PpuMI sites and the PpuMI digestion of the PIV3r-l RT-PCR product produced two bands of the expected sizes for PIV3r-l (Line 2) indicating the presence of only one PpuMI site. Secondly, Wcol digestion of the RT-PCR product from PIV3r-1.2HN (Line 5) yielded 4 bands including the 0.5 kb fragment indicative of the HN gene of HPIV2 and the Wcol digestion of the RT-PCR product from PIV3r-l (Line 6) produced the two expected fragments. M identifies the Line containing the lkb DNA sequence used as the nucleotide size markers (nt) (Life Technology). Similar results confirmed the presence of the HN insert of HPIV2 in PIV3r-lcp45.2HN. Figure 5 demonstrates that PIV3r-1.2HN expresses the HPV2 HN protein the LLC-MK2 monolayers were infected with PIV3r-1, PIV3r-1.2HN or wild type virus PIV2 / V94 at an MOI of 5. The infected monolayers were incubated at 32 ° C and were labeled with a mixture of 35S-met and 3SS-cys from 18 to 36 hours after infection. The cells were harvested and lysed, and the proteins were immunoprecipitated with an anti-HPIV2 150S1 HN mAb (Durbin et al., Virology 261: 319-330, 1999; Tsurudome et al., Virology 171: 38-48, 1989, incorporated herein by reference). The immunoprecipitated samples were denatured, separated on a SDS PAGE gel from 4% to 12% and autoradiographed (Lines: 1, PIV3r-1; 2, PIV3-1.2HN3; 3, PIV2 / V9412-6). The mAb, specific for HN of HPIV2, precipitated a protein from both HN of PIV3r-1.2 and LLC-MK2 cells infected with PIV2 / V94, but not from cells infected with PIV3r-l, with an expected size for the protein HN 86kD Kd of HPIV2 (Rydbeck et al., J. Gen. Virol. ^ 9: 931-5, 1988, incorporated herein by reference). Figure 6 depicts the location and construction of the gene unit (GU) insertions or the 3 'non-coding region (NCR) extensions of the HN gene. The nucleotide sequences and cloning sites of the single restriction enzyme of the GU and NCR insertion sites are shown in panels A and B, respectively. The acting Cs transcriptional signal sequences are indicated, that is, the signal sequences at the end of the gene (GE), intergenic (IG) and start of the gene (GS). In Panel A of Figure 6, a duplex oligonucleotide is shown specifying the HN, GE, IG and GS signal sequences as well as the unique restriction enzyme recognition sequences inserted into the introduced Stul restriction site (underlined nucleotides). ) (see Figure IB and Example I for the location of the introduced Stul site). A restriction fragment from an RSV antigenome plasmid was cloned into the Hpal site. As necessary, a short duplex oligonucleotide was inserted into the MluI site of the multiple cloning site, such that the total length of the insert conformed to the rule of six. In Panel B of Figure 6, the 3 'NCR inserts of the HN gene were cloned into the Hpal site of the indicated 32 nt multiple cloning site, which has been cloned into the Stul restriction site as described in Panel A of Figure 6. The inserted sequences were made to conform to the six rule by inserting short duplex oligonucleotides into the Afluí site at the multiple cloning site. SEQ ID NO. 5 and 6 are shown in Figure 6. Figure 7 illustrates open reading frames (ORF) in the RSV insert of 3079 bp. The six possible reading frames are shown in the RSV fragment of 3079 bp (three in each orientation; 3, 2, 1, -1, -2, -3). The short bars represent the translation start codons. The long bars represent the end codons of the translation. The 3079 bp fragment is inserted into the NCR 3 'HN (NCR ins) or between the HN and L genes as a gene unit (GU ins) in such orientation that the reading frames found by the PIV3 translation machinery correspond to -3, -2 and-1 in the figure. These reading frames contain many stop codons throughout the total length of the sequence and therefore must not produce any functional proteins. Figure 8 demonstrates that the PIV3r insert and the extension mutants contain inserts of the appropriate size. RT-PCR was performed using a pair of specific primers with PIV3 flanking the insertion site and the RT-PCR products were separated by agarose gel electrophoresis. The expected size of the RT-PCR fragment for wild-type PIV3r (also referred to as JSr) is 3497 bp and that for each of the other GU or NCR PIV3r mutants was increased in length depending on the size of the insert. Panel A represents GU insertion (ins) mutants: 1. wild type PIV3r; 2. ins GU 168r nt; 2. ins GU of 678r nt; 3. ins GU of 996r nt; 4. Ins GU of 1428r nt; 5. ins GU of 1908r nt; 6. ins GU of 3918r nt. M: the digestion products of the HindIII restriction enzyme of lambda phage DNA. The sizes of the markers of relevant size are indicated. Panel B represents the NCR insertion mutants: 1. PIV3r wild type; 2. ins NCR of 258r nt; 3. ins NCR of 972r nt; 4. ins NCR of 1404r nt; 5. ins NCR of 3126r nt; 6. ins NCR of 3894r nt. M: the digestion products of the restriction enzyme tiindIII of the lambda phage DNA. The sizes of the markers of relevant size are indicated. Figures 9A-9C present growth curves of multiple et'apas of GU and NCR insertion mutations compared to wild type HPIV3r and cp45Lr. The LLC-MK2 monolayers in the 6-well plates were infected with each of HPIV3 in triplicate at a multiplicity of infection (m.o.i.) of 0.01 and washed 4 times after removing the virus supernatant. In intervals of 0 hrs. and 24 hrs. for 6 days after infection, 0.5 ml of the virus medium from each cavity was collected and 0.5 ml of fresh medium was added to each cavity. The collected samples were stored at -80 ° C. The virus present in the samples was quantified by titration in monolayers LLC-K2 in plates of 96 cavities incubated at 32 ° C. The titers of the viruses were expressed as TCID5o / ml. The average of three independent infections of an experiment is shown. The lower limit of detection is 0.7 logioTCID5o / ml. The insertion mutants of Figure 9A-GU; the insertion mutants of Figure 9B-NCR insertion mutant of Figure 9C-cp45L / GU. Figure 10 illustrates the strategy for the placement of a supernumerary gene insert between the HPIV3r P and M genes and the NCR gene in the three (3 ') prime direction of the HPIV3r p gene was modified to contain an AflI restriction site in the positions of the antigenomic sequence 3693-3698 (Durbin, J. Virol. 74: 6821-31, 2000, incorporated herein by reference). This site was then used to insert a duplex oligonucleotide (shown in the upper part) that contains cis-acting transcriptional signal sequences of HPIV3, that is, the recurrent elements of the end of the gene (GE), integenics (IG) and start of the gene (GS). The duplex also contains a series of restriction enzyme recognition sequences available for insertion of the foreign ORFs. In the case of the HN ORFs of HPIV1 and HPIV2, the cloning sites were iVcoI and HINdIII. The insertion of a foreign ORF at the multiple cloning sites was placed under the control of a set of HPIV3 transcription signals, such that in the final recombinant virus the gene was transcribed into an mRNA separated by HPIV3 polymerase. As necessary, a short duplex oligonucleotide was inserted into the MluI site of the multiple cloning site to adjust the final length of the genome to be a multiple of six, which has been shown to be a requirement for efficient RNA replication (Calain et al., J. Virol., 67: 4822-30, 1993, Durbin et al., Virology 234: 74-83, 1997b). A similar strategy was used to place the HPIV1 and HPIV2 gene inserts between the N and P genes of HPIV3r using an AflII restriction site introduced at positions 1677-1682 9 (SEQ ID NO: 7). Figure 11 is a diagram (without scale) of the genomes of a chimeric HPIV3r series containing one or two supernumerary gene inserts, each of which codes for a protective antigen of PIV1, PIV2, or measles virus. The schematic representation of the HPIV3r (unscaled) showing the relative position of the aggregated inserts encoding the HN glycoprotein (hemagglutinin-neuraminidase) of HPIV1 (WM) or HPIV2 (ES) or HA glycoprotein (hemagglutinin) of the measles virus (EH-j) inserted in the structure HPIV3r (?). The HPIV3r construct depicted in the lower part contains an insert 3918-nt (GU) that does not code for a protein (E3) (Skiadopoulos et al., Virology 272: 225-34, 2000, incorporated herein by reference) . Each of the foreign inserts is under the control of a set of transcription signals from the start of the gene and end of the HPIV3 gene and is expressed as a separate mRNA. to. Monolayers LLC-MK2 in 6-well plates (Costar) were separately infected in triplicate in one m.o.i. of 0.01 with each of the indicated viruses. The supernatants were collected on days 5, 6 and 7 and the virus was quantified as described previously (Skiadopoulos et al., Virology 272; 225-34, 2000). The mean peak titer obtained for each virus is shown as logTCIDso / ml. b. Average of two experiments. Serially diluted viruses were incubated at 32 ° C and 39 ° C in LLC-MK2 monolayer cultures for 7 days and the presence of the virus was determined by hemadsorption with guinea pig erythrocytes. The average reduction in the titer at 39 ° C is shown compared to that of 32 ° C. Figure 12 provides a diagram (without scale) illustrating the insertion of a supernumerary gene insert into a structure of HPIV3r, HPIV3r-NB, in which the ORF N of HPIV3 is replaced by its counterpart HPIV3, which confers a phenotype of attenuation due to the host classification restriction (Bailly et al., J. Virol. 74: 3188-3195, 2000a, incorporated herein by reference). Schematic representations of HPIV3r (upper part) and biologically derived BPIV3 (lower part) are shown. The relative position of the ORF N sequence of the Kansas strain of BPIV3 (Hi) and the hemagglutinin gene of measles virus (ES) in the PIV3 structure are shown. In each case, the foreign sequence is under the control of a set of HPIV3 transcription signals. A portion of the plasmid vector containing the NgoMIV site C ~ ~) is shown. Designations are provided for the cloning of antigenomic cDNA (left) and its coded recombinant viruses (right). Figure 13 illustrates the insertion of G or F of RSV as an additional supernumerary gene in a promoter-proximal position in the genome of B / HPIV3r. B / HPIV3r is a recombinant version of BPIV3 in which the F and HN genes of BPIV3 have been replaced by their counterparts HPIV3 (FH and HNH respectively). A BIpI site was created in structure B / HPIV3 immediately downstream of the ATG start codon of the ORF N. The open reading frames (ORF) G or F of RSV were inserted into this BlpI site. The 3 rd end of any RSV insert was designed to contain the gene end (GE) and gene start (GS) sequences of PIV3 (AAGTAAGAAAAA (SEQ ID NO. 8) and AGGATTAAAG, respectively, in a positive sense) separated by the CTT intergenic sequence. Each insert also contained a Nhel site that can I serve as an insertion site for an additional supernumerary gene 5. AGGATTAAAGAACTTACCGAAAGGTAAGGGGAAAGAAATCCTAAGAGCTTAGC GATG (SEQ ID NO 9). GCTTAGCGATG (SEQ ID No. 10). AAGCTAGCGCTTAGC (SEQ ID NO.11).

GCT AGCAAAAAGCTAGCACAATG (SEQ ID No. 12). ^ .0 Figure 14 illustrates the multiclicic replication of B / HPIV3r-Gl, B / HPIV3r-Fl and its recombinant and biological precursor viruses in Simian LLC-MK2 cells. Triplicate monolayer cultures were infected at an input MOI of 0.01 TCID50 per 15 cell with B / HPIV3r-Gl, B / HPIV3r-Fl, or the following control viruses: Ka of BPIV3r, which is the recombinant version of strain Ka of BPIV3; B / HPIV3r, which is the version of BPIV3r in which the glycoprotein F and HN genes of BPIV3 were replaced with their 20 counterparts HPIV3; HPIV3 JS, which is the JS strain of HPIV3 biologically derived; and Ka of BPIV3, which is the biologically derived version of the Ka strain of BPIV3. The virus titers are shown as the mean logio TCID50 / ml of the samples in triplicate. 25 The lower limit of detection of this analysis is 10i.45TCID5o / ml í Figure 15 is a diagram (without scale) of the genomes of BPIV3r (# 1) and a series of the chimeric B / HPIV3r (# 2-6) containing substitutions of the F and HN genes of BPIV3 by those of HPIV3 (# 2) or HPIV1 (# 3-6) and one or two inserts of supernumerary genes that encode the ORF of F and / or HN of HPIV2 ( # 4-6). The schematic representation of B / HPIV3.1r chimeric viruses (unscaled) showing the relative position of the supernumerary gene coding for the F glycoprotein or HN of HPIV2 (F2 and HN2, respectively). Each foreign insert is under the control of a set of transcripts of initiation of the gene and end of the HPIV3 gene and is designed to be expressed as a separate mRNA. Figure 16 provides a diagram (without scale) illustrating the insertion of an HA coding sequence of the measles virus into several different PIV3r structures. Three structures are illustrated: HPIV3r-1 wild type (superior construction); wild type (second top construction) (Tao et al., J. Virol. 72: 2955-2961, 1998 incorporated herein by reference) in which the glycoprotein F and HN genes of HPIV3 have been replaced by those of HPIV1; and HPIV3r-lcp45L (third construct), a wild-type HPIV3r-1 derivative containing three point mutations of attenuating amino acid in the L gene derived from the cp45 vaccine strain (Skiadopoulos et al., J. Virol. 72: 1762 -8, 1998, incorporated herein by reference). The relative position of the ORF F and HN sequences of HPIV1 are shown (E3) and the HA gene of measles virus (BH) in structure PIV3r (CZ1). In each case, each foreign ORF is under the control of a set of HPIV3 transcription signals. The relative locations of the three amino acid point mutations L cp45 in the L (*) gene are indicated. A portion of the plasmid vector containing the unique NgdM.IV site is shown (Figure 17 illustrates the construction of pFLC.PIV32hc of chimeric antigenomic cDNA of PIV3-PIV2 which encodes the full-length PIV2 HN and F proteins. of cDNA containing the full-length PIV2 F ORF flanked by the indicated restriction sites (Al) was amplified from PIV2 / V94 vRNA using RT-PCR and a specific primer pair PIV2 F (1, 2 in Table 22). This fragment was digested with Ncol plus BamHI (Cl) and ligated to the Ncol-BamHI window of pLit.PIV31.fhc (Bl) to generate pLi t. PIV32 Fhc (GAVE) . In parallel, the cDNA fragment containing the full-length PIV2 HFN ORF flanked by the indicated restriction sites (A2) was amplified from PIV2 / V94 vRNA using RT-PCR and an HN specific primer pair of PIV2 ( 3, 4 in Table 22). This fragment was digested with Ncol plus HindIII (C2) and ligated to the Ncol-HindIII window of pLit.PIV31.HNhc (B2) to generate pLit. PIV32HNhc (D2). pLit.PIV32Fhc and pLit. PIV32HNhc were digested with PpuMI and Spel and assembled together to generate pLit. PIV32hc (E). pLit.PIV32hc was additionally digested with BspEI and Spel and entered into the BspEI-Spel window of p38 'APIV31hc (F) to generate p38'APIC32hc (G). The chimeric PIV3-PIV2 construct was introduced into the BspEI-Sphl window of pFLC.2G + hc to generate pFLC.PIC32hc (H). Figure 18 represents the construction of pFLC.PIV32TM and pFLC. PIV32TMcp45, full length PIV3-PIV2 chimeric antigenomic cDNA, which codes for F and HN proteins containing ectodomains derived from PIV2 and transmembrane and cytoplasmic domains derived from PIV3. The region of the ORF F PIV3, in pLit.PIV3.F3a (Al), which codes for the ectodomain was deleted (Cl) by PCR using a specific primer pair F of PIV3 (9, 10 in Table 22). The ORF F region of PIV2 coding for the ectodomain was amplified from pLit. PIV32Fhc (Bl) using PCR and a specific primer pair F of PIV2 (5, 6 in Table 22). The two resulting fragments (Cl and DI) were ligated to generate pLit. PIV32FTM (El). In parallel, the HN ORF region of PIV3, in pLit.PIV3.HN4 (A2), encoding the ectodomain was deleted (C2) by PCR using a specific primer pair HN of PIV3 (11, 12 in Table 22) . The ORF HN region of PIV2 coding for the ectodomain was amplified from pLit. PIV32HNhc (B2) by PCR and a specific primer pair HN of PIV2 (8, 9 in Table 22). Those two DNA fragments (C2 and D2) were ligated together to generate pLit.PIV32HNTM (E2). pLit. PIV32FT and pLit. PIV32HNTM were digested with PpuMI and Spel and assembled to generate pLit.PIV32TM (F). The BspEI-Spel fragment from pLit.PIV32TM was ligated to the BspEI-Spel window of p38'_PIV31hc (G) to generate p38'_PIV32TM (H). The insert containing F and HN of chimeric PIV3-PIV2 was introduced as a BspEI-Sphl 6.5 kb fragment in the BspEI-Sphl window of pFLC.2G + .hc and pFLCcp45 to generate pFLC. PIV32TM and pFLC. PIV32TMcp45 (I) respectively. Figure 19 shows the construction of pFLC. PIV32CT and pFLC. PIV32Ct cp45 of chimeric antigenomic cDNA of full-length PIV3-PIV2 coding for F and HN proteins containing an ectodomain derived from PIV2, a transmembrane domain derived from PIV2 and a cytoplasmic domain derived from PIV3. The ORF F region of PIV3 in pLit.PIV3.F3a (Al) coding for the ectodomain and the t ansmembrane domain was deleted (Cl) by PCR using a specific primer pair F of PIV3 (17, 18 in Table 22) . The ORF F region of PIV2 coding for the ectodomain plus the transmembrane domain was amplified from pLit. PIV32Fhc (Bl) using PCR and a specific primer pair F of PIV2 (13, 14 in Table 22). The two resulting fragments (Cl and DI) were ligated to generate pLit. PIV32FCT (El). In parallel, the HN ORF region of PIV3 in pLit.PIV3.HN4 (A2), which codes for the ectodomain and the transmembrane domain was deleted (C2) by PCR using a specific primer pair HN of PIV3 (19, 20 in the Table 22). The ORF HN region of PIV2 coding for the ectodomain plus the transmembrane domain was amplified from pLit. PIV32HNhc (B2) by PCR using a specific primer pair HN of PIV2 (15, 16 in Table 22). Those two DNA fragments (C2 and D2) were ligated to generate pLit. PIV32HNCT (E2). pLit. PIV32FCT and pLit. PIV32HNCT were digested with PpuMI and Spel and assembled to generate pLit.PIV32CT (F). The BspEI-Spel fragment from pLit.PIV32CT was ligated to the BspEI-Spel window of p38'_PIV31hc (G) to generate p38 '__ PIV32CT (H). The insert containing F and HN of chimeric PIV3-PIV2 was introduced as a BspEI-Sphl fragment of 6.5 kb in the BspEI-Sphl window of pFLC.2G + .hc and pFLC.cp45 to generate pFLC. PIV32CT and pFLC. PIV32CTcp45 (I) respectively. Figure 20 details the generic structures of the PIV3-PIV2 chimeric viruses and the gene binding sequences for PIV3r-2CT and PIV3r-2TM. Panel A illustrates the genetic structures of chimeric viruses PIV3r-2 (in the middle of the three diagrams) as compared to that of viruses PIV3r (upper diagram) and PIV3r-l (lower diagram). The cp45 derivatives are shown marked with arrows representing the relative positions of the cp45 mutations. For the cp45 derivatives, only the F and HN genes are different while the remaining genes remain identical, all from PIV3. From top to bottom, the three chimeric PIV3-PIV2 viruses carry decreasing amounts of the PIV3 glycoprotein genes. Note that PIV3r-2, which carries the ORF of HN and F of PIV2, could not be recovered. Panel B provides the nucleotide sequence of the junctions of the chimeric glycoprotein F and HN genes for PIV3r-2 ™ provided with the translation of the protein. The shaded portions represent the PIV2 sequences. The amino acids are numbered with respect to their positions in the corresponding wild-type glycoproteins. Three extra nucleotides are inserted into HN ™ of PIV3-PIV2 as indicated for construction according to the rule of six. Panel C shows the nucleotide sequences of the junctions of the chimeric glycoprotein F and HN genes for PIV3r-2CT, provided with the translation of the protein. The shaded portions represent the PIV2 sequences. The amino acids are numbered with respect to their positions in the corresponding wild-type glycoproteins. GE = end of the gene; 1 = intergenic; GS = start of the gene; ORF = open reading frame; TM = transmembrane domain; CT = cytoplasmic domain; * = stop codon. Figure 21 documents the multicyclic replication of the PIV3r-2 chimeric viruses compared to those of the wild type precursor viruses PIV3r / JS and PIV2 / V94. Panel A - PIV3r-2T and PIV3r-2TMcp45 viruses, together with the wild type precursor viruses PIV3r / JS and PIV2 / V94, were used to infect LLC-MK2 cells in 6-well plates, each in triplicate, at a time MOI of 0.01. All cultures were incubated at 32 ° C. After 1 hour of adsorption period, the inocula were removed and the cells were washed three times with serum free OptiMEM. The cultures were covered with 2 ml per cavity of the same medium. For plates infected with PIV3r-2TM and PIV3r-2TMcp45 / 0.5 mg / ml p-trypsin was added to each well. Aliquots of 0.5 ml extracted from each cavity were taken at 24 hour intervals for 6 days, instantaneous freezing on dry ice and stored at -80 ° C. Each aliquot was replaced with 0.5 ml of fresh medium with or without p-trypsin as indicated above. The virus present in the aliquots was titrated on LLC-NK2 plates with liquid cover at 32 ° C for 7 days and the endpoints were identified with hemadsorption. Panel B - the PIV3r-2CT and PlV3r-2CTcp45 together with the wild type precursor viruses PIV3r / JS and PIV2 / V94, were used to infect LLC-MK2 in 6-well plates, each in triplicate as described in the Panel A. The aliquots were removed and processed in the same manner as described in Panel A. Virus titers were expressed as LogioTCID50 / ml ± standard errors for the two experiments presented in Panel A and B.

PESCRIPTION OF SPECIFIC MODALITIES The present invention provides methods and compositions for the production and use of chimeric parainfluenza viruses (PIV) and associated vaccines. The chimeric viruses of the invention are infectious and immunogenic in humans and other mammals and are useful for generating immune responses against one or more of the PIV, for example against one or more of the human PIV (HPIV). Alternatively, chimeric PIVs are provided to produce an immune response against a selected PIV and one or more additional pathogens, for example, against both HPIV and measles viruses. The immune response produced may involve either or both humoral responses and / or delivered by the cell. Preferably, the chimeric PIVs of the invention are attenuated to provide a desired balance of attenuation and immunogenicity for the use of the vaccine.

In this way, the invention provides novel methods for designing and producing attenuated chimeric PIVs that are useful as vaccine agents to prevent and / or treat infection and related disease symptoms that can be attributed to PIV and other pathogens. According to the methods of the invention, chimeric parainfluenza viruses or subviral particles are constructed using a PIV "vector" antigenome or genome that is recombinantly modified to incorporate one or more antigenic determinants of a heterologous pathogen. The genome or vector antigenome consists of a partial or complete PIV genome or antigenome, which itself can incorporate nucleotide modifications such as for example attenuation mutations. The genome or vector antigenome is modified to form a chimeric structure through the incorporation of a heterologous gene or genomic segment. More specifically, the chimeric PIVs of the invention are constructed through a cDNA-based virus recovery system that provides recombinant viruses that incorporate a partial or complete vector or an "antecedent" PIV genome or antigenome combined with one or more "donor" nucleotide sequences that code for heterologous antigenic determinants. Preferably, the PIV vector comprises an HPIV genome or antigenome, although non-human PIVs, for example a bovine PIV (BPIV), can be used as a vector to incorporate the antigenic determinants of human PIVs and other human pathogens. In exemplary embodiments described herein, a human PIV3 (HPIV3) vector genome or antigenome is modified to incorporate one or more genes or genomic segments encoding antigenic determinants of one or more of the heterologous PIVs (e.g., HPIV1 and / or HPIV2), and / or a pathogen without PIV (eg, measles virus). In this way, the constructed chimeric PIVs of the invention can produce an immune response against a specific PIV, for example, HPIV1, HPIV2, and / or HPIV3, or against a pathogen without PIV. Alternatively, compositions and methods are provided for producing a polyspecific immune response against the multiple PIV, for example, HPIV1 and HPIV3, or against one or more of the HPIV and a pathogen without PIV such as, for example, measles virus. The exemplary chimeric PIV of the invention incorporates a chimeric PIV genome or antigenome as described above, as well as a major nucleocapsid (N) protein, a phosphoprotein (P) nucleocapsid and a large polymerase protein (L). Additional PIV proteins can be included in various combinations to provide a range of infectious subviral particles, to a complete viral particle or a viral particle containing supernumerary proteins, antigenic determinants or other additional components. In preferred aspects of the invention, the chimeric PIV incorporates a partial or complete human PIV vector genome or antigenome combined with one or more heterologous genes or genomic segments from a second human PIV pathogen or one without PIV such as measles virus. The "vector" PIV genome or antigenome typically acts as a receptor or carrier to which one or more "donor" genes or genomic segments of a heterologous pathogen are added or incorporated. Typically, the polynucleotides that encode one or more antigenic determinants of the heterologous pathogen are added to the genome or antigenome vector or are substituted therein to provide a chimeric PIV which thereby acquires the ability to produce an immune response in a host selected against the heterologous pathogen. In addition, the chimeric virus may exhibit other novel phenotypic characteristics as compared to one or both of the vector and heterologous PIV pathogens. The genome or partial or complete vector antigenome in general acts as a structure in which heterologous genes or genomic segments of a different pathogen are incorporated. Frequently, the heterologous pathogen is a different PIV of which one or more genes or genomic segments are combined with or replaced by the genome or antigenome vector. In addition to providing novel immunogenic characteristics, the addition or substitution of heterologous genes or genomic segments within the PIV vector strain can confer an increase or decrease in attenuation, developmental changes or other desired phenotypic changes as compared to the corresponding phenotypes of the vector unmodified and the donor viruses. Heterologous genes and genomic segments from other IVP that can be selected as inserts or additions within the chimeric PIV of the invention include genes or genomic segments that encode the N, P, C, D, V,, F, HN proteins and / or L of PIV or one or more antigenic determinants thereof. The heterologous genes or genomic segments of a PIV can be added as a supernumerary genomic element to a genome or partial or complete antigenome of a different PIV. Alternatively, one or more heterologous genes or genomic segments of a PIV can be substituted at a position corresponding to a wild-type gene order position of one of the counterpart genes or genomic segments that is deleted within the PIV vector antigen or genome. Still in additional embodiments, the heterologous genome or genomic segment is added or replaced at a position that is more promotora-proximal or promoter-distant as compared to a position of the wild-type gene of the counterpart gene or genome segment within the genome or antigenome vector for enhancing or reducing, respectively, the expression of the heterologous gene or genomic segment. The introduction of the heterologous immunogenic proteins, the protein domains and the immunogenic epitopes to produce chimeric PIV is particularly useful for generating novel immune responses in an immunized host. In addition or in substitution of an immunogenic gene or genomic segment, the donor pathogen within a genome or recipient PIV vector antigenome may generate an immune response directed against the donor pathogen, the PIV vector or both against the donor and the vector. To achieve this end, the chimeric PIV can be constructed to express a chimeric protein, for example, an immunogenic glycoprotein having a cytoplasmic tail and / or a transmembrane domain specific for a vector fused to a heterologous ectodomain of a different PIV or pathogen without PIV to provide a fusion protein that produces an immune response against the heterologous pathogen. For example, a heterologous genomic segment encoding a glycoprotein ectodomain from a human PIVI HN or F glycoprotein may be linked to a corresponding genomic segment encoding the corresponding HPIV3 cytoplasmic HN or F glycoprotein and the transmembrane domains to form a chimeric HPIV3-1 glycoprotein that produces an immune response against HPIV1. Briefly, the PIV of the invention that expresses a chimeric glycoprotein comprises a major nucleocapsid (N) protein, a phosphoprotein (P) nucleocapsid, a large polymerase protein (L), and a genome or HPIV vector antigen that is modified to encode a Chimeric glycoprotein. The chimeric glycoprotein incorporates one or more heterologous antigenic domains, fragments or epitopes of a second antigenically distinct HPIV. Preferably, this is achieved by replacement within the HPIV vector genome or antigenome of one or more heterologous genomic segments of the second HPIV that codes for one or more antigenic domains, fragments or epitopes whereby the genome or antigenome codes for the chimeric glycoprotein which is antigenically distinct from the precursor vector virus. In more detailed aspects, the heterologous genomic segment or segments preferably code for an ectodomain of glycoprotein or immunogenic portion or epitope thereof and optionally includes other portions of the heterologous or "donor" glycoprotein, e.g. both an ectodomain and a transmembrane region. which are substituted for the ecto- and transmembrane domains of glycoprotein counterpart in the genome or antigenome vector. The chimeric glycoproteins in this context can be selected from HPV HN and / or F glycoproteins, and the genome or vector antigenome can be modified to code for the multiple chimeric glycoproteins. In preferred embodiments, the HPIV vector genome or antigenome is a partial HPIV3 genome or antigenome and the second antigenically distinct HPIV is either HPIV1 or HPIV2. In an exemplary embodiment described below, both HN and F glycoprotein ectodomains of HPIV2 are substituted to correspond to the corresponding HN and F glycoprotein ectodomains in the HPIV3 vector genome or antigenome. In another exemplary embodiment, the PIV2 ectodomain and the transmembrane regions of one or both of the HN and / or F glycoproteins are fused to one or more corresponding PIV3 cytoplasmic tail regions to form the chimeric glycoprotein. Additional details relating to these aspects of the invention are provided in the United States Patent Application entitled "CONSTRUCTION AND USE OF RECOMBINANT PARAINFLUENZA VIRUSES EXPRESSING A CHIMERIC GLYCOPROTEIN", filed December 10, 1999 by Tao et al. and is identified by the Attorney File No. 17634-000340, incorporated herein by reference. To construct the chimeric PIVs of the invention that carry a heterologous antigenic determinant of a pathogen without PIV, a heterologous gene or genomic segment of the donor pathogen can be added or substituted in any functional position in the genome or antigenome vector. In one embodiment, heterologous genes or genomic segments of a pathogen without PIV can be added (i.e., without substitution) within a genome or PIV vector antigenome to create novel immunogenic properties within the resulting cloning. In these cases, the heterologous gene or genomic segment can be added as a supernumerary gene or genomic segment, optionally for the additional purpose of attenuating the resulting chimeric virus, in combination with a complete PIV vector genome or antigenome. Alternatively, the heterologous gene or genomic segment can be added together with the deletion of a • selected gene or genomic segment in the genome or antigenome vector. In preferred embodiments of the invention, the heterologous gene or genomic segment is added in an intergenic position within the genome or partial or complete PIV vector antigenome.

Alternatively / the genomic segment or gene can be inserted into other non-coding regions of the genome, for example, within the 5 'or 3"non-coding regions or at other positions where the non-coding nucleotides occur within the genome or antigenome vector. In one aspect, the heterologous gene or genomic segment is inserted into a non-coding site by overlapping a cis-acting regulatory sequence within the genome or antigenome vector, for example, within a sequence required for efficient replication, transcription and / or translation. These regions of the genome or antigenome vector represent target sites for the disruption or modification of regulatory functions associated with the introduction of the heterologous gene or genomic segment. As used herein, the term "gene" generally refers to a portion of a target genome, for example a PIV genome, that codes for an mRNA and typically begins at the 5 'end with a to the initial gene (GS) and ends at the 3 'end with the gene end signal (GE). The term "gene" can also be interchanged with the term "translational open reading frame", or ORE *, particularly in the case where a protein, such as for example PIV protein C, is expressed from an additional ORF in place of a single mRNA. In the case of the HPIV3 example, the genome is a negative sense single stranded RNA with 15462 nucleotides (nt) in length (Galinski et al., Virology 165: 499-510, 1998; Stokes et al., Virus Res. 25_: 91-103, 1992). At least eight proteins are encoded by the HPIV3 genome: the N nucleocapsid protein, the phosphoprotein P, the C and D proteins of unknown functions, the M protein matrix, the fusion F glycoprotein, the HN glycoprotein hemagglutinin-neuraminidase and the large proteins Polymerase L (Collins et al., 3rd ed., in "Fields Virology," BN Fields, DM Knipe, PM Howley, RM Chanock, JL Melnick, TP Monath, B. Roizman, and SE Straus, Eds., Vol. 1 , pp. 1205-1243, Lippincott-Raven Publishers, Philadelphia, 1996). The viral genome of all PIVs also contains extragenic, guide and tow regions, which possess all or part of the promoters required for viral replication and transcription, as well as non-coding and intergenic regions. In this way, the genetic map of PIV is represented as the guide 3 '-N-P / C / D / V-M-F-HN-L-5' trailer. Transcription starts at the 3 'end and continues through a sequential stop-start mechanism that is guided by short conserved motifs found at the gene boundaries. The end 51 of each gene contains an initial gene signal (GS), which directs the initiation of its respective mRNA. The 3 'terminal address of each gene contains a gene end motif (GE) that directs polyadenylation and termination. Sequences for the exemplary genome have been described for the JS strains of human PIV3 (accession number GenBank Z11575, incorporated herein by reference) and Washington (Galinski MS In Kingsbury, DW (Ed.), The Paramyxoviruses, pp. 537 -568, Plenum Press, New York, 1991, incorporated herein by reference), and for bovine PIV3 strain 910n (accession number GenBank D80487, incorporated herein by reference). To construct the chimeric PIVs of the invention, one or more of the PIV genes or genomic segments can be deleted, inserted or replaced in whole or in part. This means that deletions, insertions and partial or complete substitutions may include open reading frames and / or cis-acting regulatory sequences of any one or more of the PIV genes or genomic segments. By "genomic segment" it should be understood any length of continuous nucleotides, originating from the PIV genome, which could be part of an ORF, a gene, or an extragenic region or a combination thereof. When an objective genomic segment codes for an antigenic determinant, the genomic segment encodes at least one immunogenic epitope. capable of producing a humoral immune response or delivered per cell in a mammalian host. The genomic segment can also code for an immunogenic fragment or protein domain. In other aspects, the donor genomic segment can encode multiple immunogenic domains or epitopes, including recombinantly synthesized sequences comprising multiple, repeating, or different immunogenic domains or epitopes. The alternative chimeric PIV of the invention will contain protective antigenic determinants of HPIV1, HPIV2 and / or HPIV3. This is preferably achieved by the expression of one or more HN and / or F genes or genomic segments by the vector PIV, or as extra or substitute genes from the heterologous donor pathogen. In certain embodiments, a HPIV3-1 or HPIV3-2 chimeric virus can be constructed to be used as a vaccine or vector strain, in which the HN and / or F genes of HPIV1 or HPIV2 replace their PIV3 counterparts (Skiadopoulos et al., Vaccine 18 / .503-510, 1999; Tao et al., Vaccine 17: 1100-1108, 1999; U.S. Patent Application Serial No. 09 / 083,793, filed May 22, 1998 (and the International Application published as WO 98/53078); U.S. Patent Application Serial No. 09 / 458,813, filed December 10, 1999; U.S. Patent Application Serial No. 09 / 459,062, filed December 10, 1999; each incorporated herein by reference). In this context, a candidate for chimeric PIV1 vaccine has been generated using the PIV3 cDNA rescue system by replacing the PIV3 open reading frames (ORF) HN and F with those of PIV1 in a full length PIV3 cDNA. which contains the three attenuating mutations in L. The recombinant chimeric virus derived from this cDNA is designated PIV3r-l.cp45L, (Skiadopoulos et al., J. Virol. 72: 1762-8, 1998; Tao et al., J. Virol., 72: 2955-2961, 1998; Tao et al., Vaccine 17: 1100-1108, 1999, incorporated herein by reference). The PIV3r-l. cp45L is attenuated in hamsters and a high level of resistance is induced to inoculate them with PIV1. A recombinant chimeric virus, designated PIV3r-l. cp45, has also been produced to contain 12 of the 15 cp45 mutations, that is, excluding the mutations in HN and F and is quite attenuated in the upper and lower respiratory tracts of hamsters (Skiadopoulos et al., Vaccine 18: 503 -510, 1999, incorporated herein by reference). In preferred embodiments of the invention, the chimeric PIV carries one or more of the major antigenic determinants of a human PIV, or against multiple human PIVs that include HPIV1, HPIV2 or HPIV3. These preferred vaccine candidates produce an effective immune response in humans against one or more of the selected HPIVs. As noted above, antigenic determinants that produce a response against HPIV can be encoded by the genome or antigenome vector or can be inserted within or be linked to the genome or PIV vector antigenome as a heterologous gene or gene segment. The main protective antigens of human PIVs are their HN and F glycoproteins. However, all PIV genes are candidates for coding for antigenic determinants of interest, including internal protein genes that can code for those determinants, such as CTL epitopes. Preferred chimeric PIV vaccine viruses of the invention carry one or more determinants of each of a plurality of HPIV or HPIV and a pathogen without PIV. Chimeric PIV constructed in this manner includes a partial or complete HPIV genome or antigenome, for example from HPIV3 and one or more heterologous genes or genomic segments encoding antigenic determinants of a heterologous PIV, for example, HPIV1 or HPIV2. In alternative embodiments, one or more genes or genomic segments that code for one or more antigenic determinants of HPIV1 or HPIV2 can be added to the partial or complete HPIV3 genome or antigenome or can be substituted therein. In several exemplary embodiments described below, both HPIV1 genes encoding HN and F glycoproteins are substituted for the counterpart HPIV3 HN and F genes in a candidate for chimeric PIV vaccine. These and other constructs provide the chimeric PIVs that produce an immune response either mono- or poly-specific in humans for one or more HPIVs. Further detailed aspects of the invention are provided in the U.S. Patent Application entitled "CONSTRUCTION AND USE OF RECOMBINANT PARAINFLUENZA VIRUSES EXPRESSING A CHIMERIC GLYCOPROTEIN", filed December 10, 1999 by Tao et al. and identified by Attorney's file No. 17634-000340, and United States Patent Application entitled USE OF RECOMBINANT PARAINFLUENZA VIRUS (PIV) AS A VECTOR TO PROTECT AGAINST DISEASE CAUSED BY PIV AND RESPIRATORY SYNCYTIAL VIRUS (RSV), filed on December 10, 1999 by Murphy et al. and identified by the Attorney's file No. 17634-000330, each one incorporated herein by reference. In the exemplary aspects of the invention, the heterologous genes or genomic segments encoding antigenic determinants of both HPIV1 and HPIV2 are added to or incorporated into the whole or partial HPIV3 vector genome or antigenome. For example, one or more HPIV1 genes or genomic segments encoding HN and / or F glycoproteins, or antigenic determinants thereof and one or more HPIV2 genes or genomic segments encoding HN and / or F glycoproteins or antigenic determinants are they can add to the genome or partial or complete HPIV3 vector antigenome or they can be incorporated into it. In an example described below, both HPIV1 genes encoding HN and F glycoproteins are replaced by the counterpart HPIV3 HN and F genes to form a chimeric vector HPIV3-1 genome or antigenome. This construction of the vector can be further modified by the addition or incorporation of one or more genes or genomic segments that code for antigenic determinants of HPIV2. In this way, the specific constructs exemplifying the invention are provided to yield the chimeric PIVs having both HPIV1 and HPIV2 antigenic determinants, as exemplified by the candidates for PIV3r-1.2HN and PIV3r-lcp45.2HN vaccine described herein. later. In other preferred aspects of the invention, the chimeric PIV incorporates a modified HPIV vector genome or antigenome to express one or more major antigenic determinants of the pathogen without PIV, eg, measles virus. The methods of the invention can be adapted in general for incorporation of antigenic determinants from a wide range of additional pathogens within candidates for chimeric PIV vaccines. In this regard, the invention also provides for the development of candidates for vaccines against respiratory syncytial viruses (RSV), subgroup A and subgroup B. Mumps virus, human papillomavirus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filovirus, bunyavirus, flavivirus, alphavirus and influenza viruses among other pathogens. In this respect, the pathogens that may be targeted for the development of the vaccine according to the methods of the invention include viral and bacterial pathogens, as well as protozoa and multicellular pathogens. Useful antigenic determinants of many important human pathogens in this context are known or identified. easily for incorporation into the chimeric PIV of the invention. In this way, the major antigens have been identified for the above example pathogens, including the HA and F proteins of measles virus.; RSV proteins F, G, SH and M2, mumps virus HN and F proteins, human papilloma virus Ll proteins, human immunodeficiency virus type 1 or type 2 gpl60 protein, proteins gB, gC, gD, gE, gG, gH, gl, gj, gK, gL and gM of herpes simplex virus and cytomegalovirus, G protein of rabies virus, gp350 protein of Epstein Barr virus; protein G of fi'lovirus, protein G of bunyavirus, proteins E and NS1 of flavivirus and alpha virus E. These major antigens, as well as other antigens known in the art for the listed pathogens and others, are well characterized for the extent to which many of their antigenic determinants, including full-length proteins and their corresponding antigenic domains, fragments and epitopes, are identified, mapped and characterized for their respective immunogenic activities. Among the many example mapping studies, which identify and characterize the major antigens of various pathogens for use within the invention are epitope mapping studies directed to the hemagglutinin-neuraminidase (HN) gene of HPIV3 (van Wyke Coelingh et al. ., J. Virol. 6: 1: 1473-1477, 1987, incorporated herein by reference). This report provides detailed antigenic structural analyzes for 16 antigenic variants of HPIV3 variants selected by using monoclonal antibodies (MAb) for the HN protein that inhibit neuraminidase, hemagglutination or both activities. Each variant has a single point mutation in the HN gene, which codes for a simple amino acid substitution in the HN protein. The operational and topographic maps of the HN protein correlate well with the relative positions of the substitutions. The computer-assisted analysis of the HN protein predicts a secondary structure composed mainly of hydrophobic ß sheets interconnected by random hydrophilic tail structures. The HN epitopes are located in predicted tail regions. The epitopes recognized by MAbs that inhibit the neuraminidase activity of the virus are located in a region that appears to be structurally conserved among various paramyxovirus HN proteins and which may represent the sialic acid binding site of the HN molecule. This example work, which employs methods for conventional antigen mapping, identifies simple amino acids that are important for the integrity of the HN epitopes. The majority of these epitopes are located in the C-terminal half of the molecule, as expected for a protein anchored at its N-terminus (Elango et al., J. Virol. 57: 81-489, 1986). The previously published operational and topographic maps of the PIV3 HN indicated that the employed MAbs recognize six distinct epitope groups (I to VI) organized into two topographically separate sites (A and B), which are partially bridged by a third site. (C) These groups of epitopes represent useful candidates for antigenic determinants that can be incorporated, alone or in various combinations, within the chimeric PIVs of the invention. (See also, Coelingh et al., Virology 143: 569-582, 1985; Coelingh et al., Virology 162: 137-143, 1988; ay et al., Virology 148: 232-236, 1986; Rydbeck et al. , J. Gen. Virol. 67: 1531-1542, 1986, each incorporated herein by reference). Additional studies by van Wyke Coelingh et al. (J. Virol. 63: 375-382, 1989) provide additional information that relates to the selection of antigenic determinants of PIV for use within the invention. In this study, twenty-six monoclonal antibodies (MAb) (14 neutralizing and 12 non-neutralizing) are used to examine the antigenic structure, biological properties and natural variation of the fusion glycoprotein (F) of HPIV3. Analysis of selected antigenic variants in the laboratory and clinical isolates of PIV3 indicates that the MAb panel recognizes at least 20 epitopes, 14 of which participate in the neutralization. Competitive binding analyzes confirmed that 14 neutralization epitopes were organized into three major non-overlapping antigenic regions (A, B and C) and one bridge site (AB), and that the 6 non-neutralizing epitopes form four sites (D, E, F and G). The majority of neutralizing MAbs were involved in non-reciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralizing epitopes. Other antigenic determinants for use within the invention have been identified and characterized for respiratory syncytial virus (RSV). For example, Beeler et al., J. Virol. 63: 2941-2950, 1989, incorporated herein by reference, employs eighteen neutralizing monoclonal antibodies (MAbs) specific for the fusion glycoprotein of RSV strain A2 to construct a detailed topological and operational map of the epitopes involved in the neutralization. and fusion of RSV. The competitive binding analyzes identified three non-overlapping antigenic regions (A, B and C) and a bridge site (AB). Thirteen of the MAb-resistant mutants (MARM) were selected and the neutralization patterns of MAbs with any of the MARM or RSV clinical strains identified a minimum of 16 epitopes. MARMs selected with antibodies for six of the A and AB site epitopes exhibited a minor plaque phenotype, which is consistent with an alteration in a biologically active region of the F molecule. The MARM analysis also indicates that these neutralization epitopes they occupy topographically distinct but conformationally interdependent regions with unique biological and immunological properties. The antigenic variation of the F epitopes was then examined using 23 clinical isolates (18 from subgroup A and 5 from subgroup B) in cross-neutralization analysis with the 18 anti-F MAb. This analysis identified variable and nippervariable constant regions on the molecule and indicated that the antigenic variation in the neutralization epitopes of RSV glycoprotein F is the result of a non-cumulative genetic heterogeneity. Of the 16 epitopes, 8 were conserved in all or almost all of 1 out of 23 clinical isolates of subgroup A or subgroup B. These antigenic determinants, including full-length proteins and their constituent antigenic domains, fragments and epitopes, all represent useful candidates for integration into the chimeric PIV of the invention to produce novel immune responses as described above. (See also, Anderson et al., J. Infect. Dis. 151: 626-633, 1985; Coelingh et al., J. Virol. 63: 375-382, 1989; Fenner et al., Scand. J. Immunol. 24: 335-340, 1986; Fernie et al., Proc, Soc. Exp. Biol. Med. 171: 266-271, 1982; Sato et al., J. Gen. Virol. 6_6: 1397-1409, 1985; Walsh et al., J. Gen. Virol. 7: 505-513, 1996 and Olmsted et al., J. Virol. 63: 411-420, 1989, each incorporated herein by reference). To express the antigenic determinants of heterologous PIVs and pathogens without PIV,. The invention provides many human and non-human PIV vectors including bovine PIV vectors (BPIV). These vectors are easily modified according to the recombinant methods described herein to carry heterologous antigenic determinants and produce one or more humoral immune responses or delivered by specific cells against the heterologous pathogen and the vector PIV. In exemplary embodiments, one or more of the heterologous genes or genomic segments from a donor pathogen are combined with the HPIV3 vector genome or antigenome. In other exemplary embodiments, the heterologous gene or genomic segment is incorporated into a chimeric HPIV vector or antigenome vector; for example a chimeric HPIV3-1 vector genome or antigenome having one of both HPIV1 genes encoding the HN and F glycoproteins substituted by their counterpart HPIV3 HN and / or F genes. In more detailed embodiments, a transcription unit comprising an open reading frame (ORF) of the HA gene of measles virus is added to an HPIV3 vector genome or antigenome in various positions, which provides candidates for exemplary chimeric PIV / measles vaccine. PIV3r (HA HN-L), PIV3r (HA NP), cp45Lr (HA NP), PIV3r (HA PM) or cp45Lr (HA PM).

Alternatively, the chimeric PIV for use as a vaccine may incorporate one or more antigenic determinants of HPIV2, for example a HN gene of HPIV2, within a chimeric HPIV3-1 genome or antigenome vector. In other detailed embodiments of the invention, chimeric PIVs are designed to incorporate heterologous nucleotide sequences encoding respiratory syncytial virus (RSV) protective antigens to produce candidates for attenuated, infectious vaccines. The cloning of the RSV cDNA and other exposure is provided in Provisional Patent Application No. 60 / 007,083 filed on September 27, 1995; U.S. Patent Application 08 / 720,132 filed September 27, 1996; United States Provisional Patent Application No. 60 / 021,773 filed July 15, 1996; United States Provisional Patent Application No. 60 / 046,141 filed May 9, 1997; U.S. Provisional Patent Application No. 60/047, 634 filed May 23, 1997; U.S. Patent Application No. 08 / 892,403 filed July 15, 1997 (corresponding to International Application No. WO 98/02530); U.S. Patent Application No. 09/291, 894 filed April 13, 1999; International Application No. PCT / US00 / 09696, filed April 12, 2000, corresponding to United States Patent Application Serial No. 60/129, 006, filed April 13, 1999; Collins et al., Proc Nat. Acad. Sci. U.S. A. 92: 11563-11567, 1995; Bukreyev et al., J. Virol., 70: 6634-41, 1996, Juhasz et al., J. Virol. 71: 5814-5819, 1997; Durbin et al., Virology 235: 323-332, 1997; He et al. Virology 237: 249-260, 1997; Barón et al. J. Virol. 21: 1265-1271, 1997; Whitehead et al., Virology 247: 232-9, 1998a; Whitehead et al., J. Virol. 72 ^ 4467-4471, 1998b; Jin et al. Virology 251: 206-214, 1998; and Whitehead et al., J. Virol. 73: 3438-3442, 1999, and Bukreyev et al., Proc. Nat. Acad. Sci. U.S. A. 9_6: 2367-72, 1999, each incorporated herein by reference in its entirety for any purpose). Other reports and analyzes incorporated or mentioned herein identify and characterize antigenic determinants RSV that are useful within the invention. PIV chimeras that incorporate one or more RSV antigenic determinants, preferably comprise a PIV vector genome or antigenome (e.g., HPIV1, HPIV2, HPIV3) with a heterologous gene or genomic segment encoding an RSV glycoprotein, protein domain, (for example, a glycoprotein ectodomain) or one or more immunogenic epitopes. In one embodiment, one or more genes or genomic segments of the RSV F and / or G genes are combined with the vector genome or antigenome to form the candidate for chimeric PIV vaccine.

Certain of these constructs will express chimeric proteins, for example fusion proteins having a cytoplasmic tail and / or a PIV transmembrane domain fused to an SV ectodomain to provide a novel attenuated virus that produces a multivalent immune response for both PIV and RSV . As noted above, it is often desirable to adjust the phenotype of the chimeric PIV to be used as a vaccine by introducing additional mutations that increase or decrease the attenuation or otherwise alter the chimeric virus phenotype. Detailed descriptions of the materials and methods for producing recombinant PIV from cDNA and for producing and testing various mutations and nucleotide modifications shown herein are provided as further aspects of the present invention, for example, Durbin et al. Virology 235: 323-332, 1997; U.S. Patent Application Serial No. 09/083, 793, filed May 22, 1998; U.S. Patent Application Serial No. 09 / 458,813, filed December 10, 1999; U.S. Patent Application Serial No. 09 / 459,062, filed December 10, 1999; United States Provisional Application No. 60 / 047,575, filed on May 23, 1997 (corresponding to International Application No. WO 98/53078) and United States Provisional Application No. 60 / 059,385 filed on September 19 of 1997, each incorporated herein by reference. In particular, these documents describe the methods and procedures for performing mutagenesis, isolation and. characterization of the PIV to obtain attenuated mutant strains (eg, temperature sensitive (ts), cold-pass (cp) adapted to cold (ca), small-plate (sp) and mutant strains restricted to the host grade (hr)) and to identify the genetic changes that specify the attenuated phenotype. Together with these methods, the above documents detail the procedures for determining the replication immunogenicity, genetic stability and protective efficacy determinants of the biologically derived and amplified recombinantly produced human PIV in the accepted model systems, including murine and non-human primate model systems. In addition, these documents describe general methods for developing and testing immunogenic compositions, including monovalent and bivalent vaccines, for the prophylaxis and treatment of PIV infection. JL, methods for producing infectious recombinant PIV by construction and expression of cDNA encoding a PIV genome or antigenome co-expressed with essential PIV proteins are also described in previously incorporated documents, which include the description of the following example plasmids that are can be used to produce clones of infectious PIV: p3 / 7 (131) (ATCC 97990); p3 / 7 (131) 2G (ATCC 97889); and p218 (131) (ATCC 97991); each deposited under the terms of the Budapest treaty with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Virginia 20110-2209, E.U.A., and in accordance with the access numbers identified above. Also described in the above-incorporated references are methods for constructing and evaluating infectious recombinant PIV, which are modified to incorporate specific phenotype mutations identified in biologically derived PIV mutants, for example, cold-killed (cp) mutants, adapted to cold (ca), restricted to host classification (r), small plate (sp), and / or temperature sensitive (ts), for example, mutant strain cp45 JS of HPIV3. The mutations identified in these mutants can be easily incorporated into the chimeric PIVs of the current invention. In exemplary embodiments, one or more attenuating mutations occur in the polymerase L protein, for example, in a position corresponding to Tyr942, Leu992 or Thri558 of cp45 JS. Preferably, these mutations are incorporated into the chimeric PIV of the invention by an identical or conservative amino acid substitution as identified in the biological marant. In more detailed aspects, the chimeric PIV for use as a vaccine incorporates one or more mutations in. where yrg42 is replaced by His, Leug92 is replaced by Phe, and / or Thri558 is replaced by Lie. Substitutions that are conservative for these replacement amino acids are also useful to achieve the desired attenuation in candidates for chimeric vaccine. Strain cp45 JS of HPIV3 has been deposited under the terms of the Budapest treaty with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Virginia 20110-2209, E.U.A. with the Designation of Patent Deposit PTA-2419. Other example mutations that can be adopted in the chimeric PIVs from the biologically derived PIV mutants include one or more mutations in the N protein, which include specific mutations at a position corresponding to the Val96 or Ser3Q9 residues of cp45 JS. In more detailed aspects, these mutations are represented as Val96 to Ala or Ser3e9 to Ala or substitutions that are conservative thereof. The replacement of amino acids in protein C is also useful within the chimeric PIV of the invention., for example, a mutation in a position corresponding to Ile96 of cp45 JS, preferably represented by an identical or conservative substitution of Ile§6 to Thr. Additional example mutations that can be adapted from the biologically derived PIV mutants include one or more mutations in the F protein, and which include mutations adopted from cp45 JS at a position corresponding to the residues Ile42o or Ala ^ s of cpAb JS, preferably represented by substitutions of? 1? 2 acid? to Val or Ala ^ so to Thr or conservative substitutions thereof. Alternatively or in addition, the chimeric PIV of the invention may adopt one or more amino acid substitutions in the HN protein as exemplified by a mutation at the position corresponding to the Val384 residue of cp45 JS, preferably represented by the alsea substitution to Ala. Still further embodiments of the invention include chimeric PIV that incorporate one or more mutations in the non-coding portions of the PIV genome or antigenome, for example in a 3 'leader sequence, which specifies the desired phenotypic changes such as for example attenuation. Exemplary mutations in this context can be designed at a position on the 3 'leader of the chimeric virus at a position corresponding to nucleotide 23, 24, 28 or 45 of cp45 JS. Still further exemplary mutations can be designed in the start sequence of the N gene, for example by changing one or more nucleotides in the start sequence of the N gene, for example, at a position corresponding to nucleotide 62 of cp45 JS. In more detailed aspects, the chimeric PIV incorporates a T to C change in nucleotide 23, a change C to T in nucleotide 24, a change G to T in nucleotide 28 and / or a change T to A in the nucleotide 45. Additional mutations in the extragenic sequences are exemplified by a change A to T in the start sequence of the N gene at a position corresponding to nucleotide 62 of JS. These above example mutations that can be designed in a chimeric PIV of the invention have been successfully designed and recovered in recombinant PIV - as represented by the recombinant PIV clones designated cp45r, cp45r L, cp45r F, cp45r M, cp45r HN, cp45r C, cp45r F, cp45r 3'N, cp3'NLr and cp45r 3 'NCMFHN (Durbin et al., Virology 235: 323-332, 1997; Skiadopoulos et al., J. Virol. 72 ^: 1762-1768, 1998; Skiadopoulos et al., J. Virol, 73: 1374-1381, 1999; U.S. Patent Application Serial No. 09 / 083,793, filed May 22, 1998; U.S. Patent Application Serial No. 09 / 458,813, filed December 10, 1999; U.S. Patent Application Serial No. 09 / 459,062, filed December 10, 1999; U.S. Provisional Application No. 60 / 047,575, filed May 23, 1997 (corresponding to International Publication No. WO 98/53078), and United States Provisional Application No. 60 / 059,385, filed on 19 September 1997, each incorporated herein by reference). In addition, the references incorporated above describe the construction of chimeric PIV recombinants -, for example, having the HN and F genes of 'HPIV1 substituted on an antecedent HPIV3 genome or antigenome, which is further modified to carry one or more of the attenuating mutations identified in cp45 JS of HPIV3. A chimeric recombinant incorporates all attenuating mutations identified in the L-gene of cp45. It has already been shown that all cp45 mutations outside the heterologous HN and F genes (HPIV1) can be incorporated into a recombinant HPIV3-1 to provide a candidate for chimeric, attenuated vaccine. From cp45 JS and other biologically derived PIV mutants, a large "menu" of attenuating mutations is provided, each of which can be combined with any other mutations to adjust the level of attenuation, immunogenicity and genetic stability in the chimeric PIV. of the invention. In this context, many of the chimeric PIVs will include one or more, and preferably two or more, mutations from biologically derived PIV mutants, for example, any combination of mutations identified in cp45 JS. The preferred chimeric PIVs within the invention will incorporate a plurality and even a full complement of the mutations present in cpAb JS or other mutant PIV strains derived biologically. Preferably, these mutations are stabilized against reversal in the chimeric PIV by multiple nucleotide substitutions at a codon specifying each mutation. Still further mutations that can be incorporated into the chimeric PIV of the invention are mutations, eg, attenuating mutations, identified in the heterologous PIV or other non-segmented negative-strand RNA viruses. In particular, attenuation and others. Desired mutations identified in a negative-chain RNA virus can be "transferred", eg, copied to a corresponding position within the genome or antigenome of a chimeric PIV. Briefly, the desired mutations in a heterologous negative-strand RNA virus are transferred to the chimeric PIV receptor (either in the genome or antigenome vector or in the donor gene or genomic segment). This involves mapping the mutation in the heterologous mutant virus, routinely identifying the alignment of the corresponding site in the recipient PIV and performing the mutation of the natural sequence in the PIV receptor for the mutant genotype (either by an identical mutation or conservative), as described in International Application No. PCT / US00 / 09695, filed April 12, 2000, which corresponds to the United States Provisional Patent Application Serial No. 6-0 / 129, -006, filed April 13, 1999, incorporated herein by reference. As this discussion shows, it is preferred to modify the chimeric receptor PIV genome or antigenome to encode an alteration in the target mutation site that corresponds conservatively to the alteration identified in the ether-mutant virus. For example, if an amino acid substitution marks a mutation site in the mutant virus compared to the corresponding wild type sequence, then a similar substitution can be designed in the corresponding residues in the recombinant virus. Preferably, the substitution will specify an identical or conservative amino acid for the substitute residue present in the mutant viral protein. However, it is also possible to alter the natural amino acid residue at the mutation site non-conservatively with respect to the residue eμ? ^^ ?? in the mutant protein. For example, by using any other amino acid to break or impair the function of the wild type residue. Negative-chain RNA viruses of which example mutations are identified and transferred into a recombinant PIV of the invention include other PIV's (eg, HPIV1, HPIV2, HPIV3, BPIV and MPIV), RSV, Linea virus. (SeV), Newcastle disease virus (NDV), simian virus 5 (SV5), measles virus (MeV), rinderpest virus, canine distemper virus (CDV), rabies virus (RaV) and virus vesicular stomatitis (VSV), among others. A variety of exemplary mutations are set forth, among which include: an amino acid substitution of phenylalanine at position 521 of the RSV L protein which corresponds to a phenylalanine substitution and can therefore be transferred to the same (or a conservatively related amino acid) at position 456 of the L protein of HPIV3. In the case of mutations marked by deletions or insertions, these can be introduced as corresponding deletions or insertions in the recombinant virus, however, the particular size and amino acid sequence of the deleted or inserted protein fragment can vary. Attenuation mutations in biologically derived PIV and other non-segmental negative-strand RNA viruses for incorporation into the chimeric PIV of the invention can occur naturally or can be introduced into wild-type PIV strains by mutagenesis methods. well known. For example, strains of PIV precursor attenuated incompletely can be produced by chemical mutagenesis during virus development in cell cultures to which a chemical mutagen has been added, by selecting the virus that has been subjected to a passage at suboptimal temperatures with in order to introduce developmental restriction mutations, or by the selection of a mutagenized virus that produces small plaques (sp) in the cell culture, as described in the references incorporated above. By "biologically derived PIV" is meant any PIV not produced by recombinant means. In this way, the biologically derived PIV includes all naturally occurring PIVs, including, for example, a naturally occurring PIV that has a wild-type genomic sequence and PIV that has allelic or mutant genomic variations from a reference wild-type PIV sequence, for example, a PIV having a mutation that specifies an attenuated phenotype. Likewise, the biologically derived PIV includes PIV mutants derived from a precursor PIV by, among others, artificial mutagenesis and selection procedures. As noted above, the production of a sufficiently attenuated biologically attenuated PIV mutant can be carried out by various known methods. One of these methods involves subjecting a partially attenuated virus to a step in cell culture at progressively lower attenuation temperatures. For example, partially attenuated mutants are produced by passage in cell cultures at suboptimal temperatures. In this way, a cp mutant or another partially attenuated PIV strain is adapted to an efficient development at a lower temperature by passage in the culture. This selection of the mutant PIV during the cold passage practically reduces any residual virulence in the derived strains compared to the partially attenuated precursor. Alternatively, specific mutations can be introduced into the biologically derived PIV by subjecting a partially attenuated precursor virus to chemical mutagenesis, for example, to introduce ts mutations or, in the case of viruses that are already ts, additional ts mutations sufficient for confer an attenuation and / or increased stability of the ts phenotype of the attenuated derivative. The means for the introduction of ts mutations into the PIV include the. replication of the virus in the presence of a mutagen such as, for example, 5-fluorouridine according to generally known procedures. Other chemical mutagenes can also be used. The attenuation may result from a ts mutation in almost any PIV gene, although it has been found that the target particularly arranged for this purpose is the polymerase gene (L). The level of sensitivity to the replication temperature in the attenuated PIV of example for use within the invention is determined by comparing its replication at a tolerant temperature to that at various restrictive temperatures. The lowest temperature at which the replication of the virus is reduced 100 times or more compared to its replication at the tolerable temperature is called the deactivation temperature. In experimental animals and humans, both the replication and the virulence of -PIV correlates with the temperature of deactivation of the mutant. The cp45 JS mutant of HPIV3 has been found to be genetically relatively stable, fairly immunogenic and satisfactorily attenuated. The analysis of the nucleotide sequence of this biologically derived virus and of the recombinant viruses incorporating various individual and combined mutations found therein indicates that each increased attenuation level is associated with the specific nucleotide and amino acid substitutions. The references incorporated above also expose how to distinguish routinely between silent incidental mutations and those responsible for phenotypic differences - by introducing mutations, separately and in various combinations, into the genome or antigenome of clones of infectious PIV. This process is coupled with the evaluation of the phenotypic characteristics of the precursor viruses and derivatives identifies the mutations responsible for these desired characteristics such as attenuation, sensitivity to temperature, adaptation to cold, small plate size, host classification restriction, etc. Mutations thus identified are collected in a "menu" and then entered as desired, individually or in combination, to adjust the chimeric PIV of the invention to an appropriate level of attenuation, immunogenicity, genetic resistance to reversal of an attenuated phenotype, etc., as desired. According to the above description, the ability to produce infectious PIV from cDNA allows the introduction of specific designed changes within the chimeric PIV. In particular, infectious recombinant PIVs are used for the identification of specific mutations in attenuated, biologically derived PIV strains, for example mutations specifying ts, ca, att and other phenotypes. The desired mutations of this identified form are introduced into the strains for chimeric PIV vaccine. The ability to produce viruses from cDNA allows the routine incorporation of. these mutations, individually or in various selected combinations, in a clone of full-length cDNA, after the phenotypes of the rescued recombinant viruses containing the introduced mutations have already been determined. By identifying and incorporating the specific mutations associated with the desired phenotypes, for example, a cp or ts phenotype, in the cloning of infectious chimeric PIV, the invention provides other site-specific modifications in, or within a close proximity to, the identified mutation. While most of the attenuating mutations produced in biologically derived PIVs are individual nucleotide changes, other "site-specific" mutations can also be incorporated by recombinant techniques into a chimeric PIV. As used herein, site-specific mutations include insertions, substitutions, deletions or rearrangements of 1 to 3, up to about 5 to 15 or more altered nucleotides (eg, altered from a PIV sequence type). wild-type, from a sequence of a selected mutant PIV strain, or from a precursor recombinant PIV clone subjected to mutagenesis). These site-specific mutations can be incorporated in the region or within it of a selected biologically derived point mutation. Alternatively, the mutations can be introduced into several other contexts within a PIV cloning, for example in or near a cis-acting regulatory sequence or a nucleotide sequence that encodes an active protein site, binding site, epitope immunogenic, etc. Site-specific PIV mutants typically retain a desired attenuation phenotype, although they may additionally exhibit altered phenotypic characteristics unrelated to attenuation, eg, enhanced or enhanced immunogenicity, and / or improved development. Additional examples of the desired site-specific mutants include recombinant PIV designated to incorporate additional, stabilizing nucleotide mutations in a codon specifying a point attenuating mutation. Where possible, two or more nucleotide substitutions are introduced into the codons that specify the attenuation amino acid changes in a precursor mutant or recombinant PIV clone, providing a PIV with greater genetic resistance to the reversal of an attenuated phenotype. In other embodiments, site-specific nucleotide substitutions, additions, deletions or rearrangements are introduced at the 5 '(N-terminal address) or the 3' address (C-terminal address), for example, from 1 to 3, 5 a 10 and up to 15 nucleotides or more 5 'or 3', relative to a white nucleotide position, for example, to construct or remove an existing cis-acting regulatory element. In addition to single and multiple point mutations and site-specific mutations, the changes to chimeric PIV discussed herein include deletions, insertions, substitutions or rearrangements of one or more genes or genomic segments. In particular, deletions involving one or more genes or genomic segments, whose deletions have been shown to provide additional desired phenotypic effects, are useful. Thus, U.S. Patent Application Serial No. 09 / 350,821, filed by Durbin et al. on July 9, 1999, incorporated herein by reference, describes the methods and compositions by means of which the expression of one or more HPIV genes, for example, one or more of the ORF C, D and / or V, it is reduced or removed by modifying the PIV genome or antigenome to incorporate a mutation that alters the coding assignment of an initiation codon or mutations that introduce one or more codons terminators. Alternatively, one or more of the C, D and / or V ORFs may be deleted in whole or in part to render the corresponding proteins partially or completely non-functional or to decompose protein expression completely. The chimeric PIV that has these mutations in genes C, D and / or V or other non-essential, has phenotypic characteristics quite desirable for the development of the vaccine. For example, these modifications may specify one or more desired phenotypic changes that include (i) altered cell growth properties., (ii) attenuation in the upper and / or lower respiratory tract of mammals, (iii) a change in the size of the viral plaque, (iv) a change in the cytopathic effect, and (v) a change in immunogenicity. A mutant for "separation" of PIV example lacking the ORF C expression designated rC-KO, was able to introduce a protective immune response against the inoculation of wild-type HPIV3 in a non-human primate model despite its attenuation phenotype beneficial. Thus, in more detailed aspects of the current invention, the chimeric PIV incorporates the deletion or separation mutations in the ORF C, D and / or V or other non-essential gene that alters or removes the expression of the selected genes by segments genomic This can be achieved, for example, by introducing a mutation into the frame shift or the terminator codon into a selected coding sequence, altering the translational start sites, changing the position of a gene or introducing a start codon in the direction of 'to alter its rate of expression, by changing the GS and / or GE transcription signals to alter the phenotype, or to modify an RNA editing site (eg, growth, temperature restrictions or transcription, etc.). In more detailed aspects of the invention, chimeric PIVs are provided in which the expression of one or more genes, for example, C, D and / or V ORFs, are removed at the translational or transcriptional level without deletion of the gene or a segment thereof, by, for example, introducing multiple translational terminator codons into an open translational reading frame (ORF), altering a start codon or modifying an editing site. These forms of separation viruses will often exhibit reduced growth rates and small plate sizes in the tissue culture. In this way, these methods provide novel, even additional, types of attenuating mutations that remove the expression of a viral gene that is not one of the major viral protective antigens. In this context, separation virus phenotypes produced without deletion of a gene or genomic segment can alternatively be produced by deletion mutagenesis, as described, to effectively avoid the correction mutations that can restore the synthesis of a protein. White. Other gene separations for the deletion and separation mutants of the C, D and / or V ORFs can be made using alternative designs and methods that are well known in the art (as described, for example, in (Kretschmer et al., Virology 216; 309-316, 1996; Radecke et al., Virology 217: 418-421, 1996; Kato et al., EMBO J.] J6: 578-587, 1987; and Schneider et al. ., Virology 277: 314-322, 1996, each incorporated herein by reference). The nucleotide modifications that can be introduced into the chimeric PIV constructs of the invention can alter small numbers of bases (e.g., 15 to 30 bases, up to 35 to 50 bases or more), large blocks of nucleotides (e.g. 50-100, 100-300, 300-500, 500-1,000 bases), or almost complete or complete genes (for example 1,000-1,500 nucleotides, 1,500-2,500 nucleotides, 2,500-5,000 nucleotides, 5,00-6,5,000 nucleotides or more) in the genome or antigenome vector or in the heterologous donor gene or genomic segment, depending on the nature of the change (ie, a small number of bases can be changed to insert or remove an immunogenic epitope or change a small genomic segment, whereas large blocks of bases are involved when genes or large genomic segments are added, replace delete or rearrange) . In related aspects, the invention provides for the supplementation of mutations adopted in a cloning of chimeric PIV from the biologically derived PIV, for example, the cp and ts mutations, with additional types of mutations involving the same or different genes in a PIV cloning. modified additionally. Each of the PIV genes can be selectively altered in terms of expression levels, or can be added, deleted, substituted or rearranged, in whole or in part, alone or in combination with other desired modifications, to provide a PIV chimeric that exhibits novel vaccine characteristics. Thus, in addition to the combination with the attenuating mutations adopted from the biologically derived PIV mutants, the present invention also provides a range of additional methods for attenuating or otherwise modifying the phenotype of a chimeric PIV based on the recombinant design of the clones of infectious PIV. A variety of alterations can occur in an isolated polynucleotide sequence encoding a target gene or genomic segment, which includes a donor or receptor gene or genomic segment in a chimeric PIV genome or antigenome for incorporation into infectious cloning. More specifically, to achieve the desired structural and phenotypic changes in the recombinant PIV, the invention allows the introduction of modifications which delete, substitute, introduce or rearrange a selected nucleotide or nucleotide sequence from a precursor genome or antigenome, as well as mutations that suppress, substitute, introduce or rearrange the complete genes or genomic segments, within a cloning of chimeric PIV. In this way, modifications in the chimeric PIV of the invention that simply alter or withdraw the expression of a selected gene are provided, for example, by introducing a stop codon into a selected PIV coding sequence or altering its home site. translational or RNA editing site, changing the position of a PIV gene relative to an operably linked promoter, introducing a start codon in the 5 'direction to alter expression rates, modifying (eg, by changing position, altering an existing sequence or replacing an existing sequence with a heterologous sequence) the GS and / or GE transcription signals to alter the phenotype (eg, development, temperature restrictions on transcription, etc.) and various other deletions, substitutions, additions and rearrangements that specify quantitative or qualitative changes in viral replication, transcription iption of the selected genes, or translation of the selected proteins. In this context, any PIV gene or genomic segment that is not essential for development can be removed or otherwise modified in a recombinant PIV to provide the desired effects of virulence pathogenesis, immunogenicity and other phenotypic characters. As for the coding sequences, non-coding, guiding, towing and intergenic regions can be similarly suppressed, replaced or modified and their phenotypic effects already analyzed, for example, by the use of minireplicons and recombinant PIV. In addition, a variety of other genetic alterations can be produced in a PIV genome or antigenome for incorporation into a chimeric PIV, alone or together with one or more attenuating mutations adopted from a biologically derived mutant PIV, for example, to adjust the development, attenuation, immunogenicity, genetic stability or provide other advantageous structural and / or phenotypic effects. These types of additional mutations are also discussed in the references incorporated above and can be easily designed in the chimeric PIV of the invention. For example, restriction site markers are routinely introduced into chimeric PIVs to facilitate construction and manipulation of the cDNA. In addition to these changes, the order of the genes can be changed in a chimeric PIV construct, a PIV genome promoter replaced with its antigenome counterpart, the portions of the genes removed or replaced and even the genes deleted completely. Different or additional modifications can be made to the sequence to facilitate manipulations, such as, for example, the insertion of unique restriction sites in various intergenic regions or elsewhere. The untranslated gene sequences can be deleted to increase the capacity of the foreign insertion sequences. Other mutations for incorporation into the chimeric PIV constructs of the invention include mutations directed towards cis-acting signals, which can be easily identified, for example, by mutational analysis of the PIV minigenomes. For example, insertion and deletion analysis of guide and trailer and flanking sequences identifies viral promoters and transcription signals and provides a series of mutations associated with varying degrees of reduced RNA replication or transcription. Saturation mutagenesis (whereby each position in turn is modified for each of the nucleotide alternatives) of these cis-acting signals has also identified many mutations that affect RNA replication or transcription. j Any of these mutations can be inserted into an antigenome or chimeric PIV genome as described herein. The evaluation and manipulation of the trans-acting proteins and the cis-acting RNA sequences using the complete antigenome cDNA is aided by the use of PIV minigenomes as described in the references incorporated above. Additional mutations within the chimeric PIVs of the invention may also include replacement of the 3 'end of the genome with its counterpart of the antigenome, which is associated with changes in RNA replication and transcription. In an exemplary embodiment, the level of expression of specific PIV proteins, such as protective HN and / or F antigens, can be increased by replacing natural sequences with ones that have been synthetically produced and designed to be consistent with efficient translation. In this context, it has been shown that codon usage may be a major factor in the level of translation of mammalian viral proteins (Haas et al., Current Biol. 6; 315-324, 1996, incorporated herein by reference). reference). Optimization by the recombinant methods of codon usage of the mRNAs encoding the PIV HN and F proteins will provide improved expression for these genes.

In another exemplary embodiment, a sequence surrounding a translational start site (preferably including a nucleotide at position 3) of a selected PIV gene is modified, alone or in combination with the introduction of a start codon in the 5 'direction, to modulate the expression of the PIV gene by specifying the ascending or descending regulation of the translation. Alternatively, or in combination with other recombinant modifications set forth herein, the expression of the chimeric PIV gene can be modulated by altering a transcriptional GE or GE signal of any genes selected from the virus. In alternative embodiments, the levels of gene expression in a candidate for chimeric PIV vaccine are modified at the level of transcription. In one aspect, the position of a selected gene in the PIV gene map can be changed to a nearby more promoter or distant promoter position, whereby the gene will be expressed more or less efficiently, respectively. According to this aspect, modulation of expression for specific genes can be achieved by providing reductions or increases in gene expression twice, more typically four times, up to ten times or more compared to wild type levels often assisted by a Proportional decrease in expression levels for reciprocally, positionally substituted genes. These and other transpositional shifts provide the novel chimeric PIV vector virus that has attenuated phenotypes, for example, due to the decreased expression of various viral proteins involved in RNA replication, or having other desirable properties such as for example antigen expression. increased. In other embodiments, chimeric PIVs useful in vaccine formulations can be conveniently modified to adapt to antigenic derivation in circulating viruses. Typically, the modification will be in the HN and / or F proteins. A complete HN or F gene, or a genomic segment that codes for a particular immunogenic region thereof, from a strain or group of PIV, is incorporated into a cDNA of chimeric PIV genome or antigenome by replacement of a corresponding region in a recipient clone of a different PIV strain or group, or by the addition of one or more copies of the gene, such that it is represented in the multiple antigenic forms. The progeny virus produced from the cloning of modified PIV can then be used in the vaccination protocols against the emerging PIV strains. The replacement of a human PIV coding sequence or a sequence without coding (e.g., a promoter, an end of the gene, start of the gene, intergenic element or other cis-acting) with a heterologous counterpart provides the chimeric PIV that has a variety of possible attenuating effects and other phenotypic ones. In particular, host classification and other desired effects arise from the replacement of a bovine PIV protein (BPIV) or murine PIV (MPIV), protein domain, gene or imported genomic segment within a background of human PIV, wherein the bovine or murine gene does not function efficiently in a human cell, for example, of the incompatibility of the heterologous sequence or protein with a biologically interactive human PIV sequence or protein (i.e., a cooperating sequence or protein) usually with the substituted sequence or protein for viral transcription, translation, assembly, etc.) or more typically in a host classification restriction, with a cellular protein or some other aspect of the cell medium that is different between the tolerant host and less tolerant. In exemplary embodiments, bovine PIV sequences are selected for introduction into human PIV based on known aspects of the structure and function of bovine and human PIV. In more detailed aspects, the invention provides methods for attenuating candidates for chimeric PIV vaccine, based on the additional construction of chimeras between HPIV and a non-human PIV, for example HPIV3 and BPIV3 (for example / as set forth in U.S. Patent Application Serial No. 09 / 586,479, filed June 1, 2000 by Schmidt et al.; Schmidt et al., J. Virol. 74: 8922-9, 2000, each incorporated herein by reference). This method of attenuation is based on host classification effects due to the introduction of one or more genes or genomic segments of non-human PIV into a chimeric virus based on human PIV vector. For example, there are many differences in nucleotide and amino acid sequence between BPIV and HPIV, which are reflected in differences in host classification. Between HPIV3 and BPIV3 the percentage of amino acids identified for each of the following proteins is: N (86%), P (65%), M (93%), F (83%), HN (77%) and L (91%). The difference in host classification is exemplified by the fairly tolerable development of HPIV3 in rhesus monkeys, compared to the restricted replication of two different strains of BPIV3 in the same animal (van Wyke Coelingh et al., J. Infect. Dis. 157: 655-662, 1988, incorporated herein by reference). Although the basis of the host classification differences between HPIV3 and BPIV3 remains to be determined, it is likely that they will involve more than one gene and multiple amino acid difference. The involvement of multiple genes and possibly cis-acting regulatory sequences, each involving multiple amino acid or nucleotide differences, provides a very broad basis for attenuation, which has not been altered by reversion. This is in contrast to the situation with other attenuated HPIV3 viruses, in vivo, that are attenuated by one or more of the point mutations. In this case, the reversal of any individual mutation may provide a significant reacquisition of virulence or, in a case where only an individual residue specifies attenuation, full virulence reacquisition. In exemplary embodiments of the invention, the genome or antigenome vector is an HPIV3 genome or antigenome, and the heterologous gene or genomic segment is an ORF N derived from, alternatively, a Ka or SF strain of BPIV3 (which are 99% related eh the amino acid sequence). The ORF N of the background antigenome HPIV3 is replaced by the ORF N of BPIV3 counterpart - providing a cloning of novel recombinant chimeric PIV. Replacement of HPIV3 ORF N from HPIV3 with that of Ka or SF from BPIV3 results in a protein with approximately 70 amino acid differences (depending on the strain involved) from that of HPIV3 N. N is one of the most conserved proteins, and the substitution of other proteins such as for example P, individually or in combination, could result in many more amino acid differences. The involvement of multiple genes and genomic segments each conferring amino acid or multiple nucleotide differences provides a broad basis for attenuation that is quite stable for reversion. This mode of attenuation is in sharp contrast to vaccine candidates for HPIV that are attenuated by one or more point mutations, where the reversal of an individual mutation can provide significant or complete virulence reacquisition. In addition, various attenuating point mutations known in HPIV typically provide a temperature-sensitive phenotype. A problem with the attenuation associated with temperature sensitivity is that the virus can be excessively restricted for replication in the lower respiratory tract while still being attenuated in the upper respiratory tract. This is because there is a temperature gradient inside the respiratory tract, with the temperature being higher (and more restrictive) in the lower respiratory tract and lower (less restrictive) in the upper respiratory tract. The ability of an attenuated virus to replicate in the upper respiratory tract can result in complications including congestion, rhinitis, fever and otitis media. In this way, the attenuation achieved only by temperature-sensitive mutations may not be ideal. In contrast, the host classification mutations present in the chimeric PIV of the invention will not confer in most cases temperature sensitivity. Therefore, the novel method of PIV attenuation provided by these types of modifications will be more stable genetically and phenotypically and less likely to be associated with residual virulence in the upper respiratory tract compared to other known PIV vaccine candidates. The above incorporated reference discloses that HPIV3 / BPIV3 chimeric recombinants, both Ka and SF are viable and replicate as efficiently in cell cultures either as HPIV3 or BPIV3 precursor - indicating that the chimeric recombinants did not exhibit gene incompatibilities that restrict replication in vitro. This property of efficient replication in vitro is important since it allows the efficient manufacture of this biological product. As well, the HPIV3 / BPIV3 chimeric recombinants Ka and SF (designated cKa and cSF), which carry only one bovine gene, are almost equivalent to their BPIV3 precursors in the degree of restriction of host classification in the respiratory tract of the rhesus monkey. In particular, cKa and cSF viruses exhibit approximately a 60-fold or 30-fold reduction, respectively, in replication in the upper respiratory tract of rhesus monkeys compared to HPIV3 replication. Based on this finding, it is expected that other BPIV3 genes will also confer desired levels of host classification restriction within the chimeric PIV of the invention. Thus, according to the methods herein, a list of mitigating determinants will be easily identified in heterologous genes and genomic segments of BPIV and other non-human PIVs that will confer, in the appropriate combination, a desired level of host classification restriction. and immunogenicity on the chimeric PIV selected for use in the vaccine. The human-chimeric PIV for use as vectors within the present invention includes a partial or complete "background" PIV genome or antigen derived from a human or bovine PIV strain or with a pattern after the same or a subset of virus recombined with one or more heterologous genes or genomic segments of a different PIV strain or subgroup virus to form the chimeric human-bovine PIV genome or antigenome. In preferred aspects of the invention, the chimeric PIV incorporates a genome or antigenome background of partial or complete human PIV combined with one or more heterologous genes or genomic segments from a bovine PIV. The partial or complete antecedent genome or antigenome typically acts as a receptor structure or vector in which heterologous genes or genomic segments of the human or bovine PIV counterpart are imported. Heterologous genes or genomic segments of the human or bovine PIV counterpart represent "donor" genes or polynucleotides that are combined with or substituted within the parent genome or antigenome to provide a chimeric human-bovine PIV that exhibits novel phenotypic characteristics as compared to one or both of the contributing PIVs. For example, the addition or replacement of heterologous genes or genomic segments within a selected recipient PIV strain can result in an increase or decrease in attenuation, developmental changes, altered immunogenicity or other desired phenotypic changes as compared to a corresponding phenotype of the unmodified and / or donor receptor (U.S. Patent Application Serial No. 09/586, 479, filed June 1, 2000 by Schmidt et al.; Schmidt et al., J. Virol. 74: 8922-9, 2000, each incorporated herein by reference). Genes and genomic segments that can be selected for use as heterologous substitutions or additions within human-bovine chimeric PIV vectors, include genes or genomic segments that encode proteins N, P, C, D, V, M, F , SH (where appropriate), HN and / or L of PIV or portions thereof. In addition, genes and genomic segments that code for proteins without PIV, for example, an SH protein, as found in mumps and SV5 viruses, can be incorporated into a human-bovine PIV of the invention. Regulatory regions, such as the 3 'extragenic or 5' trailer regions, and the start of the gene, end-gene, intergenic regions, or 3 'or 5' non-coding regions are also useful as heterologous substitutions or additions. Certain human-bovine chimeric PIV vectors for use within the invention carry one or more of the major antigenic determinants of HPIV3 in an antecedent that is attenuated by the replacement or addition of one or more BPIV3 genes or genomic segments. The main protective antigens of PIVs are their HN and F glycoproteins, although other proteins may also contribute to a protective immune response. In certain embodiments, the antecedent genome or antigenome is an HPIV genome or antigenome, for example, an antecedent genome or antigenome HPIV3, HPIV2 or HPIV1, to which one or more BPIV genes or genomic segments are added or within which replace them, preferably from BPIV3. In an exemplary embodiment described below, an ORF of the N gene of a BPIV3 is replaced by that of an HPIV. Alternatively, the background genome or antigenome may be a BPIV genome or antigenome that is combined with one or more genes or genomic segments encoding a glycoprotein HPIV3, HPIV2 or HPIV1, glycoprotein domain or other antigenic determinant. According to the methods of the invention, any BPIV gene or genomic segment, individually or in combination with one or more of the other BPIV genes, can be combined with the HPIV sequences to give rise to a candidate for human chimeric PIV vaccine. bovine. Any HPIV, among which are included different strains of a particular HPIV serotype, for example, HPIV3 will be a reasonable acceptor to attenuate the BPIV genes. In general, the HPIV3 genes or genomic segments selected for inclusion in the human-bovine chimeric PIV to be used as a human PIV vaccine will include one or more of the HPIV protective antigens such as the HN or F glycoproteins. the invention, the chimeric human-bovine PIV carrying one or more bovine genes or genomic segments exhibits a high degree of host classification restriction, for example in the. Respiratory tract of mammalian models of human PIV infection such as in non-human primates. In exemplary embodiments, a human PIV is attenuated by viewing or replacing one or more bovine genes or genomic segments for a partial or complete human background or antigenome genome, eg, HPIV3 PIV. In one example, the HPIV3 N gene is replaced by the N gene of BPIV3 to provide a novel human-bovine chimeric PIV vector and within this vector the measles HA gene is replaced to provide a candidate for HPIV / measles vaccine, multivalent, as exemplified by the recombinant HPIV3r-NB HAP-M described below. Preferably, the degree of host classification restriction exhibited by the human-bovine chimeric PIV vectors to develop candidates for vaccine of the invention can be compared to the degree of host classification restriction exhibited by the respective precursor BPIV or "donor" strain. . Preferably, the restriction must have a true host classification phenotype, that is, it must be specific for the host in question and must not restrict the replication and preparation of the in vitro vaccine in a suitable cell line. In addition, chimeric human-bovine PIV vectors carrying one or more bovine genes or genomic segments produce a high level of resistance in hosts susceptible to PIV infection. In this way, the invention provides a new basis for the attenuation of a live virus vector to develop vaccines against PIV and other pathogens, based on host classification effects. In related aspects of the invention, the human-bovine chimeric PIV vectors comprise a BPIV receptor or structural virus that incorporates one or more of the heterologous genes encoding HPV HN and / or F glycoproteins. Alternatively, the chimeric PIV may incorporate one or more genomic segments encoding an ectodomain (and alternatively a cytoplasmic domain and / or transmembrane domain), or immunogenic epitope of the HN and / or F glycoproteins of HPIV. These proteins, domains and immunogenic epitopes are particularly useful within human-bovine chimeric PIV because they generate novel immune responses in an immunized host. In particular, the HN and F proteins, and the immunogenic domains and epitopes thereof, provide the principal protective antigens. In certain embodiments of the invention, the addition or substitution of, or within, one or more immunogenic genes or genomic segments of a subgroup or strain of human PIV for a background genome or antigenome or bovine receptor provides a recombinant chimeric virus or subviral particle capable of generating an immune response directed against the human donor virus, including one or more specific human PIV subgroups or strains, while the bovine structure confers an attenuated phenotype that makes the chimera a useful candidate for the development of the vaccine. In an exemplary embodiment, one or more human PIV glycoprotein genes, e.g., HN and / or F, are added to or removed from a partial or complete bovine genome or antigenome to provide an infectious human-bovine chimera, attenuated, that produces an immune response against anti-human PIV in a susceptible host. Within an example vector (which carries the HIV and F glycoprotein genes of HPIV3 JS in the BPIV3 background), the G and F genes of the RSV glycoprotein A are successfully inserted as the additional heterologous ORF to provide candidates for HPIV vaccine / RSV, muitivalent, exemplified by the recombinant viruses B / HPIV3r-Gl and B / HPIV3r-Fl described below. In alternative embodiments, chimeric human-bovine PIV vectors additionally incorporate a gene or genomic segment encoding a protein, immunogenic epitope or protein domain derived from multiple human PIV strains, eg, two HN or F proteins or portions thereof. immunogenic thereof each from a different HPIV, for example, HPIV1 or HPIV2. Alternatively, a glycoprotein or immunogenic determinant from a first HPIV can be provided, and a second glycoprotein or immunogenic determinant from a second HPIV can be provided by substitution without the addition of an extraglucoprotein or determinant coding for polynucleotides to the genome or antigenome The replacement or addition of HPIV glycoproteins and antigenic determinants can also be achieved by constructing a genome or antigenome that codes for a chimeric glycoprotein in the recombinant virus or subviral particle, for example, having an immunogenic epitope, an antigenic region or an antigenic region. complete ectodomain of a first HPIV fused to a cytoplasmic domain of a heterologous HPIV. For example, a heterologous genomic segment encoding a glycoprotein ectodomain derived from a HN or F glycoprotein of HPIV1 or HPIV2 may be linked to a genomic segment encoding a corresponding HPIV3 HN or F glycoprotein., cytoplasmic / ectodomain in the antecedent genome or antigenome. In alternative embodiments, a chimeric human-bovine PIV vector or genome can encode a substitute, extra or chimeric glycoprotein or antigenic determinant thereof in the recombinant virus or subviral particle, to provide a viral recombinant having glycoproteins, domains of glycoprotein or immunogenic epitopes of both human and bovine. For example, a heterologous genomic segment encoding a glycoprotein ectodomain from a human PIV HN or F glycoprotein may be linked to a corresponding genomic segment encoding a corresponding cytoplasmic / endodomain HN or F glycoprotein in the genome or antigenome antecedent. Alternatively, the human PIV HN or F glycoprotein or parts thereof may be linked to a genomic segment encoding an HN or F glycoprotein or parts thereof from another strain or PIV serotype. In combination with the phenotypic effects of host classification provided in the chimeric human-bovine PIV of the invention, it is often desired to adjust the attenuation phenotype by introducing additional mutations that increase or decrease the attenuation of the chimeric virus. Thus, in further aspects of the invention, attenuated human-bovine chimeric PIV vectors are produced, in which the chimeric genome or antigenome is further modified by introducing one or more attenuating mutations that specify an attenuation phenotype in the resulting virus or subviral particle. These may include mutations in the regulatory sequences of RNA or in the encoded proteins. These attenuation mutations can be generated de novo and tested for attenuating effects according to a rationally designed mutagenesis strategy. Alternatively, attenuating mutations can be identified in the existing biologically derived mutant PIV, and then incorporated into a chimeric human-bovine PIV of the invention. In the preferred chimeric vaccine candidates of the invention, attenuation marked by replication in the lower and / or upper respiratory tract in an accepted animal model for the replication of PIV in humans, for example hamsters or rhesus monkeys, can be reduced at least approximately twice, more often approximately 5 times, 10 times, or 20 times and preferably 50 to 100 times and up to 1,000 times or more globally (for example, as measured between 3 and 8 days after the invention), compared to the development of the corresponding wild type or mutant precursor PIV strains. Cloning of the infectious chimeric PIV vector of the invention can also be designed according to the methods and compositions set forth herein to improve immunogenicity and induce a higher level of protection than that provided by infection with a PIV pathogen or without PIV wild type precursor (i.e., the vector or heterologous donor). For example, one more epitopes, protein domains or supplemental immunogenic proteins from a heterologous strain or type of PIV, or from a pathogen without PIV such as for example measles or RSV, can be added to a chimeric PIV by changes of suitable nucleotides in the genome or chimeric antigenome. Alternatively, the chimeric PIVs of the invention can be designed to add or remove proteins, protein domains or immunogenic specific proteins (e.g. by insertion substitution or deletion of amino acids) associated with desirable or undesirable immunological reactions. Within the methods of the invention, additional genes or genomic segments can be inserted into the chimeric PIV vector or antigenome genome or approximately the same. These genes may be under common control with receptor genes, or they may be under the control of an independent set of transcription signals. In addition to genes and genomic segments that code for antigenic determinants, genes of interest in this context include genes that code for cytokines, for example, an interleukin (for example interleukin 2 (IL-2), interleukin 4 (IL-) 4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 18 (IL-18)), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy), or the stimulating factor of granulocyte / macrophage colonies (GM-CSF), as well as IL-2 to IL-18, especially IL-2, IL-6 and IL-12 and IL-18, gamma-interferon (see, for example, United States Application No. 09 / 614,285, filed July 12, 2000, which corresponds to United States Provisional Application Serial No. 60 / 143,425 filed July 13, 1999, incorporated herein as reference) . The coexpression of these additional proteins provides the ability to modify and improve the immune responses against the chimeric PIV of the invention both quantitatively and qualitatively. Deletions, insertions, substitutions and other mutations including changes of complete viral genes or genomic segments within the chimeric PIV of the invention provide fairly stable vaccine candidates, which are particularly important in the case of immunosuppressed individuals. Many of these changes will result in the attenuation of the strains for the resulting vaccine, while others will specify different types of desired phenotypic changes.

For example, accessory genes (ie, non-essential for in vitro development) are excellent candidates for coding for proteins that specifically interfere with host immunity (see, eg, Kato et al., EMBO., J. 1-6: 578 -87, 1997, incorporated herein by reference). The withdrawal of these genes in vaccine viruses is expected to reduce virulence and pathogenesis, and / or improve immunogenicity. The introduction of the mutations defined above into a chimeric, infectious PIV clone can be achieved by a variety of well-known methods. By "infectious cloning" with respect to DNA it is to be understood that the cDNA or its product, or otherwise synthetic, can be transcribed into genomic or antigenomic RNA capable of serving as a template for producing the genome of an infectious virus or subviral particles. In this way, defined mutations can be introduced by conventional techniques (e.g., site-directed mutagenesis) into a cDNA copy of the genome or antigenome. The use of genomic cDNA antigenome or subfragments to collect a complete antigenome or genomic cDNA as described herein has the advantage that each region can be manipulated separately (the smaller cDNAs are easier to manipulate than the larger ones) and then they are easily collected in a complete cDNA. In that way, the complete antigenome or genomic cDNA, or any subfragment thereof, can be used as the template for the ^ directed oligonucleotide mutagenesis. This can be through the intermediary of a single-chain phagemid form, such as that used by the Muta-gene® team from Bio-Rad Laboratories (Richmond, CA) or a method using a double-stranded plasmid directly as a mold such as the equipment for 10 mutagenesis of Stratagene Chameleon (La Jolla CA), or i by the polymerase chain reaction using either an oligonucleotide primer or template containing the mutations of interest. A mutated subfragment can then be assembled in the 15 complete genome or genomic cDNA. A variety of other mutagenesis techniques are known and available for use in the production of the mutations of interest in the PIV antigenome or genomic cDNA. Mutations may vary from changes in 20 simple nucleotides replacing large pieces of cDNA that contain one or more genes or genomic regions. Thus, in an illustrative embodiment the mutations are introduced using the 25 kit for in vitro mutagenesis of the phagemid mutagen-gene available from Bio-Rad. Briefly, cDNA encoding a portion of a PIV genome or antigenome is cloned into plasmid pTZ18U, and used to transform CJ236 cells (Life Technologies, Gaithersburg, MD). The phagemid preparations are made as recommended by the manufacturer. Oligonucleotides are designed for mutagenesis by introducing an altered nucleotide at the desired position of the genome or antigenome. The plasmid containing the genome or genetically altered antigenomic fragment is then amplified and the mutated piece is then reintroduced into the full-length genome or antigenomic clone. The invention also provides methods for producing infectious chimeric PIV from one or more isolated polynucleotides, for example, one or more of the cDNAs. According to the present invention, the cDNA encoding a PIV genome or antigenome is constructed for intracellular or in vitro coexpression with the viral proteins necessary to form infectious PIV. By "PIV antigenome" is meant a polynucleotide molecule in the positive sense, isolated, which serves as the template for the synthesis of the genom.¾ PIV progeny. Preferably, a cDNA is constructed to be a positive-sense version of the PIV genome, which corresponds to the replicating intermediate RNA, or antigenome, to minimize the ability to hybridize with the positive sense transcripts of the complementary sequences coding for the proteins needed to generate a transcription nucleocapsid, replicating, that is, the sequences coding for the N, P and L proteins. For the purposes of the present invention, the genome or antigenome of the recombinant PIV of the invention only needs to contain those genes or portions thereof necessary to make that the coded viral or subviral particles are therefore infectious. In addition, genes or portions thereof can be provided by more than one polynucleotide molecule, i.e., a gene can be provided by complementing or the like of a nucleotide molecule separately, or can be expressed directly from the genomic cDNA or antigenomic. By recombinant PIV it is to be understood a viral or subviral particle of PIV or similar to PIV derived directly or indirectly from a recombinant or propagated expression system of the virus or subviral particles produced thereof. The recombinant expression system will employ a recombinant expression vector comprising an operably linked transcriptional unit comprising an assembly of at least one genetic element or elements having a regulating function of PIV gene expression, eg, a promoter, a sequence structural or coding that is transcribed into the PIV RNA and the appropriate transcription initiation and termination sequences. To produce the infectious PIV from the genome or antigenome expressed by cDNA, the genome or antigenome is coexpressed with those PIV proteins necessary to (i) produce a nucleocapsid capable of replication by RNA and (ii) make the nucleocapsid progeny competent for a RNA replication and transcription. Transcription by the genomic nucleocapsid provides the other PIV proteins and initiates a productive infection. Alternatively, the additional PIV proteins necessary for a productive infection can be delivered by coexpression. The infectious PIV of the invention is produced by intracellular or cell-free co-expression of one or more isolated polynucleotide molecules encoding a PIV genome or antigenomic RNA together with one or more polynucleotides that encode viral proteins necessary to generate a transcription nucleocapsid , replication. Among the viral proteins useful for co-expression to provide infectious PIV are the core nucleocapsid protein (N), the nucleocapsid phosphoprotein (P), the large polymerase protein (L), the fusion protein (F), the glycoprotein hemagglutinin-neuraminidase (HN) and the matrix protein (M). The products of the ORF C, D and V of the PIV are also useful in this context. The cDNAs encoding the PIV genome or antigenome are constructed for intracellular or in vitro coexpression with the viral proteins necessary to form infectious PIV. By "PIV antigenome" is meant an isolated positive-sense polynucleotide molecule that serves as a template for the synthesis of the PIV progenie genome. Preferably, a cDNA is constructed to be a positive sense version of the PIV genome corresponding to the replicating intermediate RNA, or antigenome, to minimize the possibility of hybridizing with the positive sense transcripts of the complementary sequences coding for the proteins. necessary to generate a transcription, nucleocapsid replication. In some embodiments of the invention, the genome or antigenome of a recombinant PIV (PIVr) need only contain those genes or portions thereof necessary to make the infectious viral or subviral particles encoded therein. In addition, genes or portions thereof can be provided by more than one polynucleotide molecule, i.e., a gene can be provided by the complementation or the like of a separate nucleotide molecule. In other modalities, the PIV genome or antigenome codes for all the functions necessary for the development, replication and viral infection without the participation of an auxiliary virus or viral function provided by a plasmid or an auxiliary cell line. By "recombinant PIV" is meant a viral or subviral particle of PIV or similar to PIV derived directly or indirectly from a recombinant or propagated expression system of the virus or subviral particles produced therefrom. The recombinant expression system will employ a recombinant expression vector comprising a functionally linked transcriptional unit comprising an assembly of at least one element or genetic elements that have a regulatory function in the expression of the PIV gene eg, a promoter, a structural sequence or coding that is transcribed into the PIV RNA and suitable transcription initiation and termination sequences. To produce infectious PIV from a PIV genome or PIV antigenome expressed by cDNA, the genome or antigenome is coexpressed with those PIV N, P and L proteins needed to (i) produce a nucleocapsid capable of replication by RNA and (ii) ) make progeny nucleocapsids competent for both RNA replication and transcription. Transcription by the genomic nucleocapsid provides the other PIV proteins and initiates a productive infection.

Alternatively, the additional PIV proteins necessary for a productive infection can be administered by coexpression. The synthesis of the PIV antigenome or genome together with the viral proteins mentioned above can be achieved in vitro (cell free), for example, using a combined transcription-translation reaction, followed by transfection in the cells. Alternatively, the antigenome or genomic RNA can be synthesized in vitro and transfected into cells expressing PIV proteins. In certain embodiments of the invention, complementary sequences encoding the proteins necessary to generate a PIV nucleocapsid of transcription, replication are provided by one or more of the helper viruses. These helper viruses can be wild type or mutant. Preferably, the helper virus can be phenotypically distinguished from the virus encoded by the PIV cDNA. For example, it is desirable to provide monoclonal antibodies that react immunologically with the helper virus but not with the virus encoded by the PIV cDNA. These antibodies can be neutralizing antibodies. In some embodiments, the antibodies can be used in affinity chromatography to separate the helper virus from the recombinant virus. To assist in obtaining these antibodies, mutations can be introduced into the PIV cDNA to provide an antigenic diversity of the helper virus, such as, for example, in the HN or F glycoprotein genes. In alternative embodiments of the invention, the PIV N, P, L and other desired proteins are encoded by one or more of the non-viral expression vectors, which may be the same or be separated from that which codes for the genome or antigenome. Additional proteins may be included as desired, each encoded by its own vector or by a vector encoding one or more of the NIV, P, L and other desired proteins or the complete genome or antigenome. The expression of the genome or antigenome and the proteins from the transfected plasmids can be achieved, for example, by each cDNA that is under the control of a T7 RNA polymerase promoter which in turn is supplied by infection, transfection or transduction with a expression system for the T7 RNA polymerase for example, a recombinant of the vaccinia virus MVA strain expressing the T7 RNA polymerase (Wyatt et al., Virology 210: 202-205, 1995, incorporated herein by reference in its entirety ). Viral proteins and / or T7 RNA polymerase can also be provided by transformed mammalian cells or by transfection of the preformed mRNA or protein.

A PIV antigenome can be constructed for use in the present invention by, for example, the assembly of the cloned cDNA segments, which together represent the antigenome, by the polymerase chain reaction or the like (PCR, described in, for example). US Pat. Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, San Diego, 1990, each incorporated herein by reference in its totality) of the reverse transcribed copies of the PIV mRNA or genomic RNA. For example, a first construct comprising the cDNAs containing the left end of the antigenome extending from a suitable promoter (e.g., the T7 RNA polymerase promoter) is generated and assembled into a suitable expression vector, such as for example a plasmid, cosmid, phage or vector of DNA virus. The vector can be modified by mutagenesis and / or synthetic polylinker insertion containing unique restriction sites designated to facilitate binding. For easy preparation, PIV N, P, L and other desired proteins can be assembled into one or more vectors separately. The right end of the antigenomic plasmid may contain additional sequences as desired, such as, for example, a flanking ribozome of T7 transcriptional terminators in tandem. The ribozome can be hammerhead type (for example Grosfeld et al., J. Virol. 69: 5677-5686, 1995), which could provide a 3 'end containing an individual non-viral nucleotide or can be any of the other suitable ribozymes such as for example those of hepatitis delta virus (Perrotta et al., Nature 350: 434-436, 1991), incorporated herein by reference in its entirety) that could provide a free 3 'end of nucleotides without PIV. The left and right ends are then joined by a common restriction site. A variety of insertions, deletions and nucleotide rearrangements can be made in the PIV genome or antigenome during or after the construction of the cDNA. For example, the desired, specific nucleotide sequences can be synthesized and inserted into the appropriate regions in the cDNA using the convenient restriction enzyme sites. Alternatively, these techniques such as site-specific mutagenesis, alanine scanning, PCR mutagenesis, and other such well-known techniques in this field can be used to introduce mutations in the cDNA. Alternative means for constructing a cDNA encoding the genome or antigenome include reverse transcription PCR using improved PCR conditions (eg, as described in Cheng et al., Proc. Nati. Acad. Sci. USA 91: 5695-5699 , 1994), incorporated herein by reference) to reduce the number of subunit cDNA components to less than one or two pieces. In other modalities different promoters can be used (for example T3, SP6) or different ribozomas (for example, those of the hepatitis delta virus). Different DNA vectors (e.g., cosmids) can be used for propagation to better accommodate the larger-sized genome or antigenome. Isolated polynucleotides (e.g. cDNA) encoding the genome or antigenome can be inserted into suitable host cells by transfection, electroporation, mechanical insertion, transduction or the like, into cells that are capable of supporting a productive PIV infection, e.g. , HEp-2, FRhL-DBS2, LLC-MK2, MRC-5 and Vero cells. Transfection of isolated polynucleotide sequences can be introduced into cultured cells by, for example, transfection supplied by calcium phosphate (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics] _: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J 1: 841-845, 1982), transfection delivered by DEAE dextran (Ausubel et al., Ed., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), transfection supplied by cationic lipid (Hawley. -Nelson et al., Focus l_5: 73-79, 1993) or a commercially available transfection reagent, for example, LipofectACE® (Life Technologies) or the like (each of the above references is incorporated herein by reference in As noted above, in some embodiments of the invention, the PIV and other desired NIV, P, and L proteins are encoded with one or more of the auxiliary viruses that are phenotypically detectable from which the genome is encoded. or antigenome.The PIV N, P, L and other desired proteins can also be encoded icar with one or more expression vectors which may be the same or be separated from those encoding the genome or antigenome and various combinations thereof. Additional proteins may be included as desired, encoded by their own vector or by a vector encoding one or more of the PIV N, P, L and other desired proteins, or the entire genome or antigenome. To provide infectious clones of the PIV, the invention allows a wide range of alterations to be produced recombinantly within the PIV genome (or antigenome), which provides defined mutations that specify desired phenotypic changes. By "infectious cloning" is meant a cDNA or its product, synthetic or otherwise, an RNA capable of being incorporated directly into the infectious virions that can be transcribed into the genomic or antigenomic RNA capable of serving as a template to produce the genome of infectious viral or subviral particles. As noted above, defined mutations can be introduced by a variety of conventional techniques (e.g., site-directed mutagenesis) into a cDNA copy of the genome or antigenome. The use of genomic or antigenomic cDNA subfragments to assemble a complete genomic or antigenomic cDNA as described herein has the advantage that each region can be manipulated separately, where small cDNA subjects are provided for better ease of manipulation than the older cDNA subjects and then easily congregate into a complete cDNA. In this way, the complete antigenomic or genomic cDNA to a selected subfragment thereof can be used as a template for oligonucleotide-directed mutagenesis. This can be through intermediary of a single chain phagemid form, such as that used by the MUTA-gen® equipment of Bio-Rad Laboratories (Richmond, CA), or a method using the double-stranded plasmid directly as a template such as that of the Chameleon® mutagenesis kit from Strategene (La Jolla, CA), or by the polymerase chain reaction using either an oligonucleotide primer or a template containing the mutations of interest. A mutated subfragment can then be assembled into the complete antigenome or the genomic cDNA. A variety of other mutagenesis techniques are known and can be routinely adapted for use in the production of mutations of interest in a PIV antigenome or a genomic cDNA of the invention. In this way, in an illustrative embodiment, mutations are introduced using the MUTA-gene® phagemid in the in vitro mutagenesis kit available from Bio-Rad Laboratories. In summary, the cDNA encoding a PIV genome or antigenome is cloned into the plasmid pTZ18U, and used to transform the CJ236 cells (Life Technologies). Phagemid preparations are produced as recommended by the manufacturer. Oligonucleotides are designed for mutagenesis by the introduction of a nucleotide. altered in the desired position of genome or antigenome. The plasmid containing the genome or genetically altered antigenome is then amplified. Mutations may vary from individual nucleotide changes for the introduction, deletion or replacement of large cDNA segments that contain one or more genes or genomic segments. The genomic segments may correspond to the structural and / or functional domains. For example, cytoplasmic, transmembrane or protein ectodomains, active sites such as sites that provide binding or other biomechanical interactions with different proteins, epitopic sites, for example, sites that stimulate immunosuppressed responses by binding antibodies and / or humoral or cells, etc. The genomic segments useful in this regard vary from about 15 to 35 nucleotides in the case of genomic segments that code for small protein functional domains, eg, epitopic sites, at about 50, 75, 100, 200-500 and 500-1,500. or more nucleotides. The ability to introduce mutations defined in infectious PIV has many applications, including pathogenic PIV manipulation and immunogenic mechanisms. For example, the functions of the PIV proteins, which include the N, P, M, F, HN and L proteins and the ORF products of C, D and V, can be manipulated by introducing the mutations that remove or reduce the level of the expression of proteins or that provide a mutant protein. Various structural features of genomic RNA such as promoters intergenic regions and transcription signals within the methods and compositions of the invention can also be routinely manipulated. The effects of trans-acting proteins and cis-acting RNA sequences can be easily determined, for example, by using a complete antigenomic cDNA in parallel analysis. employs PIV minigenomes (Dimock et al., J. Virol. 7: 2772-8, 1993, incorporated herein by reference in its entirety), whose rescue-dependent status is useful in the characterization of those mutants that may be too inhibitory to be recovered in the replication-independent infectious virus. Certain substitutions, insertions, deletions or rearrangements of the genes or genomic segments within the recombinant PIV of the invention can be made, (e.g., substitutions of a genomic segment encoding a selected protein or protein region, e.g. a tail cytoplasmic, transmembrane domain or ectodomain, an epitopic site or region, a binding site or region, an active site or region containing an active site, etc.) in the structural or functional relationship for an existing "counterpart" gene or genomic segment from the same or a different PIV or another source. These modifications provide the novel recombinants that have the desired phenotypic changes compared to the strains of. Wild type PIV or precursor or other viral strains. For example, recombinants of this type can express a chimeric protein having a cytoplasmic tail and / or transmembrane domain of a PIV fused to an ectodomain of another PIV. Other exemplary combinations of this type express duplicate protein regions, such as duplicate immunogenic regions. In the sense in which the present one is used, the "counterpart" genes, genomic segments, proteins or protein regions, typically come from heterologous sources (for example from different PIV genes or representing the same gene or genomic segment., ie homologous or allelic) in different types or strains of PIV. Typical counterparts selected in this context share general structural characteristics, for example, each counterpart can encode a comparable protein or protein structural domain, such as a cytoplasmic domain, transmembrane domain, ectodomain, site or region of attachment, site or epitopic region , etc. Counterpart domains and their coding genomic segments encompass a congregation of species having a range of size and sequence variations defined by a common biological activity between the domain or genomic segment variant. The counterpart genes and genomic segments, as well as other polynucleotides disclosed herein for the production of recombinant PIV within the invention, often share a substantial sequence identity with a "reference sequence" of polynucleotide selected, for example, from other Selected counterpart sequences. As used herein, a "reference sequence" is a defined sequence used as a basis for sequence comparison, eg, a segment of a cDNA or full-length gene, or a cDNA or sequence of complete gene. In general, a reference sequence is at least 20 nucleotides in length, often at least 25 nucleotides in length and often at least 50 nucleotides in length. Because the polynucleotides may each (1) comprise a sequence (ie, a portion of the complete polynucleotide) that is similar between the two polynucleotides, and (2) may additionally comprise a sequence that is divergent between the two polynucleotides, the Sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides with respect to a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" in the sense in which it is used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions in which a polynucleotide sequence can be compared to a reference sequence of at least 20. continuous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., openings) or 20 percent or less as compared for the reference sequence (which does not comprise additions or deletions) for an optimal alignment of the two sequences. The optimal alignment of the sequence to align a comparison window can be driven by the Smith & amp; s local homology algorithm; aterman, (Adv. Appl. Math. 2: 482, 1981), by the homology alignment algorithm of Needleman & unsch, (J. Mol. Biol., 48: 443, 1970), by the search for the Pearson & Lipman (Proc. Nati. Acad. Sci. USA 85: 2444, 1988) (each of which is incorporated as a reference), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package. 7.0, Genetics Computer Group, 575 StIience Dr., Madison, WI, incorporated herein by reference), or by inspection, and the best alignment is selected (i.e., that which results in the highest percentage of sequence similarity with respect to the comparison window) generated by the various methods. The term "sequence identity" means that two polynucleotide sequences are identical (ie, on a nucleotide-by-nucleotide basis) with respect to the comparison window. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences on the comparison window, which determines the number of positions at which the nucleic acid base is identical (eg, A, T, C, G, U or I) is presented in both sequences to provide the number of positions matched by the total number of positions in the comparison window., (For example, the size of the window) and by multiplying the result by 100 to provide the percentage of sequence identity. The term "substantial identity", in the sense in which it is used herein, denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence containing at least 85 percent sequence identity, preferably at less 90 percent to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence with respect to a comparison window of at least 20 nucleotide positions, with frequency, with respect to a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence with the polynucleotide sequence which may include deletions or additions with a total of 20 percent or less than the reference sequence with respect to the comparison window. The reference sequence may be a larger sequence subset. In addition to these polynucleotide sequence ratios, the proteins and protein regions encoded by the recombinant PIV of the invention are also typically selected to have conservative ratios, i.e., to have a substantial sequence identity or sequence similarity, with the selected reference polypeptides. As applied for polypeptides, the term "sequence identity" means peptides that share identical amino acids at the corresponding positions. The term "sequence similarity" means peptides having identical or similar amino acids. { that is, conservative substitutions) in the corresponding positions. The term "substantial sequence identity" means that two peptide sequences when they are optimally aligned, such as by the GAP or BESTFIT programs using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity more preferably at least 95 percent sequence identity or more, (eg, 99 percent sequence identity). The term, "substantial similarity" means that two peptide sequences carry corresponding percentages of sequence similarity. Preferably, the residual positions that are not identical differ by the conservative amino acid substitutions. Conservative amino acid substitutions refer to the exchange capacity of residues that have similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine.; a group of amino acids that have aliphatic-hydroxyl side chains is serine and threonine; A group of amino acids having side chains containing amide is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; A group of amino acids that has basic side chains is lysine, arginine and istidine; and a group of amino acids having side chains containing sulfur are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. Abbreviations for the twenty naturally occurring amino acids used herein follow conventional use (Immunology - A Synthesis, 2nd ed., ES Golub &DR Gren, eds., Sinauer Associates, Sunderland, MA, 1991, incorporated in the present as a reference). Stereoisomers (eg, D amino acids) of the twenty conventional amino acids, non-natural amino acids such as for example a, disubstituted amino acids, N-alkyl amino acids, lactic acid and other non-conventional amino acids may also be suitable components for polypeptides of the present invention. Examples of non-conventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, e- ?, N, N-trimethyllysine, e-α-acetyllysine, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine , O-methylarginine and other similar and imino acid amino acids (e.g., 4-hydroxyproline). In addition, amino acids can be modified by glycosylation, phosphorylation and the like. In order to select the viruses for candidate vaccine according to the invention, the viability, attenuation and immunogenicity criteria are determined according to well-known methods. Viruses that will be the most desired in the vaccines of the invention, must maintain viability, must have a stable attenuation phenotype, must exhibit a replication in an immunized host (albeit at lower levels) and must efficiently produce the response immune to a vaccine sufficient to confer protection against serious diseases caused by subsequent infection of the wild type virus. The recombinant PIV of the invention is not only viable and more adequately attenuated than previous vaccine candidates, although it is more genetically stable in vivo - it retains the ability to stimulate a protective immune response and in some cases to extend the protection provided by modifications multiple, for example, to induce protection against different viral strains or subgroups, or protection by a different immunological basis, for example, secretory against serum immunoglobulin, cellular immunity and the like. The recombinant PIV of the invention can be tested in various well-known and generally accepted in vitro and in vivo models to confirm adequate attenuation, resistance to phenotypic reversion and immunogenicity for use in vaccines. In in vitro analyzes, the modified virus (eg, a multiple attenuated PIV, biologically derived or recombinant) is tested, for example, for the temperature sensitivity of virus replication, ie ts phenotype and for small plate or other desired phenotype. The modified viruses are further tested in animal models of PIV infection. A variety of animal models has been described and summarized in various references incorporated herein. Model systems for PIV, including rodents and non-human primates, to evaluate the immunogenic attenuation activity of candidates for PIV vaccine are widely accepted in the art, and the data obtained from them correlate well with PIV infection. attenuation and immunogenicity in humans. According to the above description, the invention also provides compositions for use in the recombinant and infectious PIV vaccine, isolated. The attenuated virus that is a component of a vaccine is in an isolated and typically purified form. By "isolated" it is to be understood that it refers to the PIV which is in an environment other than the wild type of a wild type virus, such as the nasopharynx of an infected individual. More generally, it should be understood that "isolated" includes the attenuated virus as a component of a cell culture or a different artificial environment where it can be propagated and characterized in a controlled setting. For example, the attenuated PIV of the invention can be produced by an infected cell culture, separated from the cell culture and added to a stabilizer. For use in vaccines, the recombinant PIV produced according to the present invention can be used directly in the 1T9 formulations vaccine, or it can be lyophilized, as desired, using lyophilization protocols well known to the skilled artisan. The lyophilized virus will normally remain at approximately 4 C. When ready for use, the lyophilized virus is reconstituted in a stabilizing solution, eg, saline or comprising SPG, Mg ++ and HEPES, with or without adjuvant as will be further described below. . The PIV vaccines of the invention contain as an active ingredient an immunogenically effective amount of PIV produced as described herein. The modified virus can be introduced into a host with a physiologically acceptable carrier and / or adjuvant. Useful carriers are well known in the art and include, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparations will be combined with a sterile solution before administration as mentioned above. The compositions may contain pharmaceutically acceptable auxiliary substances as required for the approximate physiological conditions, such as adjusting the pH and buffering agents, tonicity adjusting agents, wetting agents and the like, for example / sodium acetate, sodium lactate, chloride of sodium, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate and the like. Acceptable adjuvants include incomplete Freund's adjuvant MPLMR (3-o-deacylated lipido A monophosphoryl, RIBI ImmunoChem Research, Inc., Hamilton, MT) and IL-12 (Genetics Institute, Cambridge MA), among many other adjuvants well known in the art. technique. At the time of immunization with a PIV composition as described herein, via aerosol, gout, oral, topical or other, the host's immune system responds to the vaccine by producing antibodies specific for PIV proteins, for example. F and HN glycoproteins. As a result of vaccination with an immunogenically effective amount of the PIV produced as described herein, the host becomes at least partially or completely immune to PIV infection, or resistant to the moderate development of severe PIV infection, particularly of the lower respiratory tract. The host to which the vaccines are administered can be any mammal that is susceptible to infection by PIV or a closely related virus and whose host is capable of generating a protective immune response to the antigens of the vaccination strain. Accordingly, the invention provides methods for creating vaccines for a variety of human and veterinary uses. The compositions of the vaccine containing the PIV of the invention are administered to a host susceptible to an infection by PIV or otherwise at risk thereof to enhance the immune response capabilities of the host itself. This amount is defined to be an "immunogenically effective dose". In this use, the precise amount of PIV that will be administered within an effective dose will depend on the state of health and weight of the host, the mode of administration, the nature of the formulation, etc., although in general it will vary from about 103 and 107 plaque forming units (PFU) or more than one virus per host, more commonly from approximately 10 * to 106 PFU of virus per host, in any case, vaccine formulations must provide a quantity of PIV modified of the invention sufficient to effectively protect the host patient against severe or life-threatening PIV infection. The PIV produced according to the present invention can be combined from other PIV serotypes or strains to achieve protection against multiple serotypes or PIV strains. Alternatively, protection against multiple serotypes and PIV strains can be achieved by combining protective epitopes of multiple serotypes or strains designed into a virus, as described herein. Normally, when the different viruses are administered, they will be in a mixture and will be administered simultaneously, although they can also be administered separately. Immunization with a strain can protect against different strains of the same or different serotypes. In some cases it may be desirable to combine the PIV vaccines of the invention with vaccines that induce protective responses for other agents, in particular other childhood viruses. In another aspect of the invention, PIV can be used as a vector for protective antigens of other pathogens, such as respiratory syncytial virus (RSV) or measles virus, by incorporating the sequences coding for those protective antigens in the genome. or PIV antigenome that is used to produce the infectious PIV, as described herein. In all subjects, the precise amount of the recombinant PIV vaccine administered, and the time and repeat administration, will be determined based on the patient's health and weight, the mode of administration, the nature of the formulation, etc. Doses in general will vary from about 103 to 101 plaque forming units (PFU) or more than one virus, per patient, most commonly from about 10 4 to 10 6 PFU of virus per patient. In any case, the vaccine formulations should provide a sufficient attenuated PIV amount to effectively stimulate or induce an anti-PIV immune response, for example, as can be determined by complementary fixation, plaque neutralization and / or linked immunosorbent assay. above, among other methods. In this regard, individuals are also monitored for signs and symptoms of upper respiratory disease. As with administration to chimpanzees, the vaccine attenuating virus develops in nasopharynx vaccines at levels approximately 10 times or more lower than the wild type virus, or approximately 10 times or more lower when compared to the levels of the vaccine. PIV attenuated incompletely. In neonates and infants, multiple administration may be required to produce sufficient levels of immunity. Administration should begin within the first month of life and at intervals throughout childhood, such as two months, six months, one year and two years, as necessary to maintain sufficient levels of protection against infection by natural PIV ( wild type). Similarly, adults who are particularly susceptible to infection by repeated or severe PIV, such as, for example, health care workers, day care workers, family members of young children, elderly individuals with compromised cardiopulmonary function, they may require many immunizations to establish and / or maintain immune protective responses. The levels of induced immunity can be monitored by measuring the amounts of neutralizing and secretory serum antibodies and the doses can be adjusted or vaccinations can be repeated as necessary to maintain the desired levels of protection. In addition, different vaccine viruses can be indicated by the administration of different receptor groups. For example, a PIV strain designed to express a cytokine or an additional protein rich in T cell epitopes may be particularly advantageous for adults rather than infants. PIV vaccines produced according to the present invention can be combined with viruses expressing antigens from another subgroup or strain of PIV to achieve protection against multiples. subgroups or strains of PIV. Alternatively, the vaccine virus may incorporate protective epitopes from multiple strains or subgroups of PIV designed in a PIV cloning., as described herein.

The PIV vaccines of the invention produce an immune response that is protective against severe lower respiratory tract disease, such as pneumonia and bronchiolitis when the individual is subsequently infected with the wild type PIV. While the naturally circulating virus is still capable of causing infection, particularly in the upper respiratory tract, there is a greatly reduced possibility of rhinitis as a result of vaccination and the possible increase in resistance by subsequent infection by virus-type. wild. After vaccination, there are detectable levels of serum and secretory antibodies generated in the host that are capable of neutralizing homologous wild type viruses (of the same subgroup) in vitro and in vivo. In many cases, host antibodies will also neutralize wild type virus from a different subgroup without a vaccine. Preferred PIV vaccine candidates of the invention exhibit a fairly substantial decrease in virulence compared to the wild type virus that is naturally circulating in humans. The virus is sufficiently attenuated in such a way that the symptoms of infection will not occur in the majority of the immunized individuals. In some cases, the attenuated virus may still be able to spread to unvaccinated individuals. However, its virulence is sufficiently neutralized in such a way that serious infections in the lower respiratory tract in the vaccinated or incidental host do not occur. The level of attenuation of candidates for PIV vaccine can be determined by, for example, quantifying the amount of virus present in the respiratory tract of an immunized host and by comparing the amount produced by wild-type PIV or other attenuated PIV. that have evaluated as strains for candidate vaccine. For example, the attenuated virus of the invention will have a higher degree of replication restriction in the upper respiratory tract of a fairly susceptible host, such as for example a chimpanzee, compared to the replication levels of the wild type virus, for example from 10 to 1000 times less. In order to further reduce the development of rhinorrhea, which is associated with the replication of the virus in the upper respiratory tract, a candidate virus for viral vaccine must exhibit a restricted level of replication in both the upper and lower respiratory tracts. However, the attenuated viruses of the invention must be sufficiently infectious and immunogenic in humans to confer protection in vaccinated individuals. Methods to determine the PIV levels in the nasopharynx of an infected host are well known in the literature. The levels of induced immunity, provided by the vaccines of the invention can also be monitored by measuring the amounts of neutralizing and secretory serum antibodies. Based on these measurements, the vaccine doses can be adjusted or vaccinations can be repeated as necessary to maintain the desired levels of protection. In addition, different vaccine viruses may be advantageous for different receptor groups, for example, a PIV strain designed, which expresses an additional protein rich in T cell epitopes may be particularly advantageous for adults instead of infants. In yet another aspect of the invention, PIV is employed as a vector for transient gene therapy of the respiratory tract. According to this embodiment, the recombinant PIV genome or antigenome incorporates a sequence that is capable of coding for a gene product of interest. The gene product of interest is under the control of the same or a promoter different from that which controls the expression of PIV. Infectious PIV caused by the coexpression of the recombinant PIV genome or antigenome with N proteins, P, L of PIV and other desired, and containing a sequence encoding the product of the gene of interest, is administered to a patient. The administration is usually by spray, spray or other topical application to the respiratory tract of the patient to be treated. The recombinant PIV is administered in an amount sufficient to result in the expression of therapeutic or prophylactic levels of the desired gene product. The representative gene products that can be administered with this method of preference are suitable for transient expression, including, for example interleukin-2, interleukin-4, gamma-interferon, GM-CSF, G-CSF, erythropoietin and other cytokines , glucocerebrosidase, phenylalanine hydroxylase, transmembrane conductance regulator for cystic fibrosis, (CFTR), hypoxanthine-guanine phosphoribosyl transferase, cytotoxins, tumor suppressor genes, antisense RNAs and vaccine antigens. The following examples are provided by way of illustration, without limitation. These examples document the construction of representative chimeric PIVs carrying one or more heterologous antigenic determinants according to the methods described above. In one example, the HA gene of the measles virus is inserted as an extra gene into one of the three gene linkages of a wild-type or attenuated JS strain of HPIV3, namely, the N / P, P / M binding or HN / L and recombinant chimeric viruses are recovered. The insertion of the measles HA gene at three different positions in the HPIV3 genome illustrates the range of constructs useful for transferring the antigenic determinants of foreign pathogens in the PIV vectors. Furthermore, it is expected that inserted gene units that are closer to the 3 'guide will be transcribed and expressed at higher levels than the same gene units located more distantly, which will allow a closer modulation of heterologous gene expression. (Collins et al., 3rd ed. In "Fields Virology", B. N. Fields, DM Knipe, PM Howley, RM Chanock, JL Melnick, TP Monath, B. Roizman, and SE Straus, Eds., Vol. 1 , pp. 1205-1243, Lippincott-RavenPublishers, Philadelphia, 1996). The chimeric HPIVr carrying the HA insert of the measles virus in a wild type HPIV3r background was efficiently applied in vitro although it was restricted in the replication in hamster compared to the HPIV3r virus from which it was derived. Similarly, the recombinant chimeric HPIV3 that cleaves the HA insert of the measles virus in an attenuated HPIV3r background replicated in vitro and in hamster at a level that was also slightly lower than that of the attenuated HPIV3rcp45L mutant virus from which it was derived. The amount of the HA protein expressed by the cells infected with the HA recombinants of the HPIV3r-measles virus attenuated with the HA gene in the N / P or P / M junctions was very high and even exceeded that observed in the cells infected with the virus. of native measles. The level of replication of HPIV3rcp45L with a HA insert of measles virus at the N / P or P / M junction was 10 times lower in the upper respiratory tract of the hamster than that of the HPIV3r-cp45L precursor virus indicating that the inserts of gene can inexplicably contribute to the attenuation of an HPIV3 vector. These results that identify a unique host classification phenotype are unexpected. Importantly, the hamster infection with each recombinant chimeric virus tested induced high levels of antibody for both HPIV3 and for the measles virus. The animals immunized with the attenuated chimeric chimeric HPIV3 carrying the HA insert were quite resistant to the replication of the HPIV3 inoculation virus. While the wild-type measles virus does not replicate efficiently in hamster and thus can not be used in an inoculation study, the protective efficacy of the attenuated chimeric recombinant vaccine is easily evident from the high levels of neutralization induced by the antibody. These levels are associated with a high level of measles resistance in humans (Chen et al., J. Infect, Dis. 162: 1036-42, 1990).

It is further demonstrated in the examples that attenuated chimeric recombinant HPIV vectors, which combine a structure of HPIV3 and one or more antigenic determinants of HPIV1 can also be used as vectors to express additional foreign antigens (eg from HPIV2 or a virus without PIV). This aspect of the invention takes advantage of the efficient development and excellent attenuation properties of the HPIV3 structure to carry the antigenic determinants of the multiple heterologous pathogens, as exemplified by HPIV1 and HPIV2. The cDNA encoding PIV3r-1 (a major antigen of HPIV1 carrying the non-attenuated recombinant) or PIV3r-lcp45 (a major antigen of HPIV1 carrying the attenuated recombinant) was modified by the insertion of a gene unit containing the HN gene of HPIV2 between the gene units containing the ORFs of F and HN of HPIV1. The recombinant chimeric viruses, designated PIV3r-1.2HN and PIV3r-lcp45.2HN, were easily recovered and replicated efficiently in tissue culture. Each virus exhibited a level of sensitivity to in vitro replication temperature similar to that of its precursor virus PIV3r-l or PIV3r-lcp45. The insertion of attenuated PIV2 HN for the viruses of both PIV3r-l and PIV3r-cp45 in hamster, a finding similar to that observed with the insertion of the HA measles viruses in the JSr and in the PIV3rcp45. Infection of the hamsters with these antigenic PIV3r-1 recombinants carrying the HN gene insert of PIV2 induced reactive serum antibody responses against both HPIV1 and HPIV2. In this way, it is possible to use a candidate for HPIV3r or attenuated HPIV3r-l vaccine as a vector to infect the respiratory tract of susceptible hosts and thereby induce a vigorous antibody response to the expressed foreign protective antigens of an extra gene unit , as well as against the HPIV vector itself. The presence of three HPIV antigenic serotypes, which do not provide significant cross protection, allows an efficient, sequential immunization of human infants with antigenically distinct HPIV variants each carrying the same or different heterologous antigenic determinants, eg, a protective antigen , an antigenic domain or epitope of the measles virus of one or more different viral or microviral pathogens. Sequential immunization allows the development of a primary immune response for the foreign protein, which is increased during subsequent infections with an antigenically distinct, secondary HPIV carrying one or more of the heterologous antigenic determinants, eg, a protective antigen, a antigenic or epitope domain of the "measles virus or of one or more different viral or microbial pathogens." Thus, the immunity induced for an HPIV vector can be circumvented by increasing with an antigenically distinct HPIV vector.In this con, successful immunization of animals that are immune to PIV3 has been achieved with candidates for attenuated PIV3-1 vaccine, which confirms the feasibility of sequential immunization with serotype-distinct PIV viruses even if these PIV share proteins other than HN and F (Tao et al. ., Vaccine 17: 1100-8, 1999.) In this study, the immunogenicity and efficacy of PIV3r-L. cp45L against Inoculation by PIV1 was examined in hamsters with and without previous immunity for PIV3. Hamsters efficiently infected with PIV3r-l. cp45L previously infected with the wild-type or attenuated PIV3, although there was approximately a five-fold reduction in the replication of the PIV3r-1 virus. cp45L in animals immune to PIV3. However, immunization with PIV3r-l. cp45L of the animals immune to PIV3 induced a vigorous serum antibody response to PIV1 and reduced the replication of the inoculated virus of PIV1 1000 times in the lower respiratory tract and 200 times in the upper respiratory tract These results demonstrate that the recombinant chimeric PIV3r-l .cp45L candidate vaccine can induce immunity to PIV1 even in animals immune to PIV3. This establishes the feasibility of using a sequential immunization program in which a PIV3r-l. recombinant chimeric cp45L or other PIV vaccine virus is provided following a live attenuated PIV3 vaccine. Because the PIV3r-l. cp45L easily induced a protective immunity against itself, this could also induce an effective immune response for any vectorized protective antigen that it is carrying. Also, PIV and RSV have the unusual property of being able to reinfect the respiratory tract, although reinfections are typically not associated with serious disease. Thus, the vector-based vaccine constructs of the invention are useful for increasing immune responses by a second, third or fourth administration of the same HPIV vector or by the sequential use of different vectors. In preferred sequential vaccination methods of the invention, it is desirable to sequentially immunize an infant with different PIV vectors each expressing the same heterologous antigenic determinant as, for example, the HA of measles virus. This sequential immunization allows the induction of the high titer of the antibody for the heterologous protein that is characteristic of the secondary antibody response. In one embodiment, younger infants (e.g., infants 2 to 4 months old) are immunized with an attenuated chimeric HPIV3 that expresses a heterologous antigenic determinant, e.g., the HA protein of measles virus and is also adapted for produce an immune response against HPIV3. A candidate for example vaccine useful in this context is the recombinant cp45Lr (HA P-M). Subsequently, for example, at four months of age, the infant is immunized again although with a construction of secondary PIV vector, different antigenically different from the first. A candidate for exemplary vaccine in this context is the cp45L virus of PIV3r-1 which expresses the HA gene of the measles virus and the antigenic determinants of HPIV1 as essential and functional glycoproteins of the vector. After the first vaccination, the vaccine will produce a primary antibody response for both the PIV3 HN and F proteins and for the measles virus HA protein, but not for the HN and F protein of PIV1. At the time of secondary immunization with the pv45L of PIV3r-l expressing the measles virus HA, the vaccine will be easily infected with the vaccine due to the absence of the antibody to the HN and F proteins of PIV1 and both will develop a response of primary antibody for the HN and F protective antigens of PIV1 and a high secondary antibody response titrated to the HA protein of the heterologous measles virus. A similar sequential immunization scheme can be developed where immunity occurs sequentially against HPIV3 and then against HPIV2 by one or more of the chimeric vaccine viruses described herein, concurrent with the stimulation of a protective initial high titre response and then secondary to measles or another pathogen without PIV. This strategy of sequential immunization, preferably using different serotypes of PIV as primary and secondary vectors, effectively avoids. immunity that is induced for the primary vector, a factor ultimately limiting the utility of vectors with only one serotype. In addition, according to this aspect of the invention, the example coordinated vaccination protocols can incorporate two, three, four and up to six or more viruses for chimeric HPIV vaccine separately administered simultaneously (eg, in a mixture of polyspecific vaccine). ) in a primary vaccination step, for example, to one, two or four months of age. For example, two or more and up to a total panel of HPIV-based vaccine viruses can be administered to separately express one or more antigenic determinants (ie, whole antigens, immunogenic domains or epitopes) selected from the G protein. of subgroup A of RSV, F protein of subgroup A of RSV, protein G of subgroup B of RSV, protein F of subgroup B of RSV, protein HA of measles virus and / or protein F of measles virus . Administration of the coordinated reinforcement of these same constructs for PIV3-based vaccine can be repeated at two months of age. Later, for example at four months of age, a separate panel of 2 to 6 or more of the viruses can be administered for live attenuated HPIV vaccine, antigenically distinct (with reference to the antigenic specificity of the vector) in a step of secondary vaccination. For example, secondary vaccination may include concurrent administration of a mixture or multiple formulations containing multiple constructs for HPIV3-1 vaccine that collectively express G of RSV of subgroup A, F of RSV of subgroup A, F of RSV of subgroup B , G of RSV of subgroup B, HA of measles virus and / or F of measles virus, or antigenic determinants of any combination of these proteins. This secondary immunization provides a reinforcement in the immunity of each of the RSV virus proteins and of heterotary measles or antigenic determinants thereof. At six months of age, a tertiary vaccination step involving the administration of one to six or more vaccine recombinants based on the PIV3-2 vector attenuated in vivo, separately, can be administered co-ordinated to be expressed separately or collectively G of RSV of subgroup A, F of RSV of subgroup A, G of RSV of subgroup B, F of RSV of subgroup B, HA of measles virus and / or F of measles virus or antigenic determinants thereof. Optionally, in this step, in the vaccination protocol, the PIV3r and PIV3r-1 vaccines can be administered in booster formulations. Thus, the important immunity characteristic of the secondary antibody for PIV1, PIV2, PIV3, A of RSV, B RSV and the measles viruses are all induced within the first six months of childhood. This coordinated / sequential immunization strategy, which is able to induce secondary antibody responses for multiple viral respiratory pathogens, provides a very powerful and extremely flexible immunization regime that addresses the need to immunize against each of these three viruses of PIV and other pathogens in early childhood. In other aspects of the invention, the insertion of the heterologous nucleotide sequences in the candidates for HPIV vaccine are used separately to modulate the level of attenuation of the recombinants of the candidate vaccine, for example, to the upper respiratory tract. In this way, it is possible to insert the nucleotide sequences into an HPIVr that both directs the expression of a foreign protein and that attenuates the virus in an animal host, or to use nucleotide inserts separately to attenuate the viruses for candidate vaccine. To define some of these rules that control the effect of gene insertion on attenuation, gene units of variable lengths were inserted into a wild-type HPIV3 structure and the effects of gene unit length on attenuation were examined . These novel gene unit insertions were designed not to contain a significant ORF that allows an evaluation of the effect of the unit length of the gene independently of an effect of the expressed protein of that gene. These heterologous sequences were inserted as an extra-gene unit of sizes between 168 nt and 3918 nt between the HN and L genes. In addition, the control cDNA constructs and the viruses in which the inserts of similar sizes were placed in the 3 'non-coding region of the HN gene and therefore do not include the addition of an extra gene. These viruses were produced to evaluate the effect of an increase in the total genome length and on the number of gene on attenuation. The insertion of an extra gene unit is expected to decrease the transcription of the genes in the 3 'direction of the insertion site that will affect both the total abundance and the proportions of the expressed proteins. As demonstrated herein, gene insertions or extensions greater than about 3000 nts in length attenuated the wild type virus for the upper and lower respiratory tracts of hamsters. Gene insertions of approximately 2000 nts in length further attenuated the candidate for HPIV3rcp45L vaccine for the upper respiratory tract. In summary, gene insertions can have the dual effect of both attenuating a virus for candidate vaccine and inducing a protective effect against a second virus. Extensions of the gene in the 3 'non-coding region of a gene, which can not express additional proteins, can also be attenuating in / and the same. Within these methods of the invention, the insertion length of the gene is an attenuation determinant. The GU and NCR inserts within the recombinant PIV of the invention produce an attenuation phenotype characterized by efficient in vitro replication and decreased replication in vivo, a phenotype not previously described for other paramyxovirus inserts. The attenuation mechanism that results from the GU insert may originate from one or more of the following factors that act predominantly in vivo. The addition of an extra gene unit can decrease the level of transcription of the genes in the 3 'direction because there is a transcriptional gradient in which nearby promoter genes are transcribed at a higher rate than that of the more distant promoter genes. . The diminished expression of the gene products in the 3 'direction resulting from the decreased abundance of their mRNAs could result in attenuation if their gene product is limiting or if a specific proportion of the gene products is required for an efficient replication is altered. It is considered that the transcription gradient is. a consequence of the complex detachment of the template transcriptase during transcription as well as during transfer through the gene junctions. Alternatively, the increase in the total length of the genome and the extra transcribed mRNAs can increase the level of the double-stranded viral RNA produced which in turn can induce a high level of the antiviral activity of the interferon system. Finally, the total level of genome replication can be reduced due to the increased length of the genome and the antigenome. This may arise from a decoupling of the replicase complexes from the template during the replication of the genomic RNA or antigenomic RNA. The decreased amount of the genome available for packaging in virions may result in a decrease in virus yield that results in attenuation.

The attenuation mechanism that results from an NCR insert can arise from one or more of the following factors. The extra length of the 3 'end of the HN mRNA resulting from the insertion of NCR may contribute to the instability of the mRNA and lead to a decrease in the expression of the HN protein. Alternatively, the increase in the total length of the genome and the extra length of the HN mRNA can increase the level of the double-stranded viral RNA produced which can induce a higher level of antiviral activity of the interferon system. Alternatively or additionally, the total level of genome replication can be reduced due to the increase in the length of the genome and the antigenome. This may arise from a separation of the replicase complexes from the template during the replication of the genomic RNA or antigenomic RNA. This decreased amount of genome available for packaging in virions could result in a decrease in virus yield resulting in attenuation. Finally, the addition of extra nucleotides to the 3 'end of the HN gene could decrease the level of transcription of the genes in the 3' direction because the transcriptase complex could detach the template during the transcription of the extra nucleotides at the 3 'end. 'of the HN gene. The in vitro and in vivo growth properties of the GU and NCR inserts in PIV3 are different from the previous findings with other RNA viruses in the negative sense, single chain, cited above. The insertions tested above examined expressed proteins, whereby the effect independent of the length of the insertions on viral growth in vivo can not be determined. The findings herein demonstrate that GU and NCR insertions greater than 3 kb specify an attenuation phenotype that is independent of the expressed protein. Minor inserts, for example, greater than about 2 kb, specify an additional attenuation in a partially attenuated receiver. Also, unexpectedly, the GU and NCR insertions specify a restricted replication in vivo in the absence of in vitro restricted replication. In addition, the in vivo attenuation phenotype is observed when the insertion is either in the form of a GU insert or an NCR insert - other documented insertions are in the form of only GU. Thus, the attenuation of specific in vivo replication by a GU or NCR insert that does not code for a protein represents a unique way to attenuate the members of the in vivo Monomegavirales.

EXAMPLE I Construction of the cDNAs encoding a chimeric HPIV3 HA antigenome / measles virus and the recovery of the infectious virus The full-length cDNA clones, p3 / 7 (131) 2G +, which encode the complete nucleotide antigenome 15462 of wild type virus JS PIV3, and pFLCcp45L coding for the antigenome of wild type derivative JS containing three temperature sensitive mutations specific to cp45 in the ORF L of PIV3, have been described previously (Durbin et al., Virology 235; 323-332, 1997a, Skiadopoulos et al., J. Virol. 72: 1762-8, 1998, each incorporated herein by reference). These clones were used as vectors for the insertion of the HA gene of measles virus to create both wild type and attenuated HPIV3 chimeric constructs expressing a heterologous antigenic determinant, exemplified by the HA protein of measles virus. The size of each insert containing the measles HA gene was a multiple of six such that the chimeric virus retrieved from the cDNA could conform to the six rule (Durbin et al., Virology 234; 74-83, 1997b, incorporated in the present as a reference).

Construction of the full-length chimeric HPIV3 cDNAs encoding the measles virus HA protein at the N / P or P / M junctions The Pmll for BamHI of p3 / 7 (131) 2G + (nt 1215-3903 of the PIV3 antigenome ) was subcloned into the plasmid pUC119. { pUC119 (PjnlI-BainHI)} that had been modified to include a Pmll site in the multiple cloning region. Two independent single-chain mutagenesis was performed in pUC119 (Pml I-BamHI) using the Kunkel method (Kunkel et al., Methods Enzymol.; 367-382, 1987, incorporated herein by reference); the first reaction introduced an A // II site in the 3 'non-coding region (3') of the N gene by mutation of the CTAAAT sequence in nts 1677-1682 of the antigenome for CTTAAG fpA // II NP), and the second reaction separately introduced an A // II site in the 3 'non-coding region of the P gene by mutation of the TCAATC sequence in nts 3693-3698 of the antigenome for CTTAAG (IpA // II PM). The ORF HA of the Edmonston strain of measles virus was amplified from Edmonston wild type virus by reverse transcription polymerase chain reaction (RT-PCR). The nt sequence of the open reading frame (ORF) of wild-type Edmonston HA is in Access GenBank # U03669, incorporated herein by reference (note that this sequence is the ORF only without the 3 nts in the 3 'direction or the final codon). The measles virus RNA was purified from the clarified medium using TRIzol-LS (Life Technologies, Gaithersburg, MD) followed by the procedure recommended by the manufacturer. RT-PCR was performed with the Advantage-RT equipment for PCR and Advantage-HF PCR (Clontech, Palo Alto, CA) following the recommended protocols. Primers were used to generate a PCR fragment that extends the complete ORF of the HA gene of measles virus flanked by the non-coding sequence PIV3 and the A // II restriction sites. The direct primer 5'-TTAATCTTAAGAATATACAAATAAGAAAAACTTAGGATTAAAGAGCGATGTCA CCACAACGAGACCGGATAAATGCCTTCTAC-3 '(SEQ ID NO: 13), codes for a 5'-direction of the A // II site (in italics) of the PIV3 non-coding sequence derived from the N / gene gene binding P - nts 3699-3731 (underlined), which contains the sequences GE, IG and GS (Figure 1A) and the beginning of the ORF HA of measles (in bold) preceded by the three nts of the virus without HPIV3, without measles designated in the primer. The reverse primer 5'-ATTATTGCrrAAGGTTTGTTCGGTGTCGTTTCTTTGTTGGATCCTATCTGCGA TTGGTTCCATCTTC-3 '(SEQ ID NO.14) codes for a 3' direction at the A // II site (in italics) (in the positive sense complement) of the non-coding sequence of PIV3 derived from the P gene, nt 3594-3623 (underlined) and the end of the measles HA ORF (in bold). The resulting PCR fragment was then digested with A / V1 and cloned into p. { A // IIN-P) and p (A // IIP-M) to create pUC119 (HA N-P) and pUC119 (HA P-M), respectively. PUC119 (HA N-P) and pUC119 (HA P-M) were sequenced on the complete A // II insert using a reaction ready to sequence the terminator cycle of Rhodamine d. { ABI prism, PE Applied Biosystems, Foster City, CA) and it was confirmed that the sequence was correct. The Pmll for the BamRI fragments of pUC119 (HA NP) and pUC119 (HA PM) were cloned separately into the plasmid p3 / 7 (131) 2G + of full-length antigenome cDNA as described previously (Durbin et al., Virology 235; 323-332, 1997a, incorporated herein by reference) to create pFLC (HA NP) and pFLC (HA PM) (Figure 1). The Xhol-NgcMI fragment (nt 7437-15929) from pFLCcp45L was then cloned into the XhoJ-iVgoMI window of both pFLC (HA NP) and pFLC (HA PM) and pFLCcp45L (HA NP) and pFLCcp45L (HA PM) to create pFLCcp45L and PIV3 cp45. pFLCcp45L codes for the three amino acid changes in the Pp3 cp45 L gene (position aa 942, 992 and 1558) that confers most of the temperature sensitivity and attenuation of candidate virus for cp45 vaccine (Skiadopoulos et al., J Virol. 72: 1762-8, | 1998, incorporated herein by reference) and the transfer of the Xhol-NgcMl fragment transferred those mutations.

Construction of the full-length HPIV3 chimeric cDNAs encoding the measles HA protein at the HN / L junction The HPIV3 chimeric cDNA was constructed by PCR to include a heterologous polynucleotide sequence, exemplified by the HA gene of measles virus , which codes for a heterologous antigenic determinant of the measles virus, flanked by the transcription signal and the non-coding regions of the HN gene of HPIV3. This cDNA was designed to be combined with a PI3r vector as an extra gene after the HN gene. First, Kunkel mutagenesis was used (Kunkel et al., Methods Enzymol, 154: 367-382, 1987, incorporated herein by reference), a Stul site was introduced into the 3 'non-coding region of the HN gene by the mutation of the AGACAA sequence in nts 8598-8603 of the antigenome for AGGCCT providing the plasmid p3 / 7 (131) 2G-Stu (Figure IB). A cDNA containing the measles HA ORF flanked by the HPIV3 sequences (see Figure IB) was then constructed in all three pieces by PCR. The first PCR synthesized the piece of the gene in the 5 'direction on the left. The direct primer 5'-GACAATAGGCCTAAAAGGGAAATATAAAAAACTTAGGAGTAAAGTTACGCAAT CC-3 '. (SEQ ID NO: 15) contains a Stul site (in italics) followed by the HPIV3 sequence (underlined) which includes the end in the 3 'direction of the HN gene. { HPIV3 nts 8602-8620), an intergenic region and the start signal of the gene and the sequence of the end in the 5 'direction of the HN (HPIV3 nt 6733-6753). The reverse primer 5'-GTAGAACGCGTTTATCCGGTCTCGTTGTGGTGACATCTCGAATTTGGATTTGT CTATTGGGTCCTTCC-3 '(SEQ ID No. 16) contains an Afluí site (in italics) in the 3' direction of the beginning of the Measles HA ORF (in bold) followed by the complement for Nos. 6744-6805. of HPIV3 (underlined), which are part of the non-coding region HN in the 5 'direction. The Miul site present in the ORF of the introduced measles virus was created by changing nt 27 of T (in the Edmonston wild type HA gene) to C and nt 30 of C to G. Both of these changes are not coding in the ORF of the measles virus. PCR was performed using p3 / 7 (131) 2G-Stu as templates. The resulting product, termed PCR fragment 1, was flanked by a Stul site at the 5 'end and an AfluI site at the 3' end and contains the first 36 nt of the measles HA ORF in the 3 'direction of the non-sequence. encoding the HN gene of HPIV3. The second PCR reaction synthesized the right end of the HN gene. The direct primer GTAGAACGCG-TTTATCCGGTCTCGTTGTGGTGACATCTCGAATTTGGATTTGT CTATTGGGTCCTTCC-3 '(SEQ ID NO.16) contains the Xmal (in italics) and the end of the HA ORF of measles (in bold), followed by HPIV3 nts 8525-8566 (underlined) which represents part of the untranslated region in the 3 'direction of the HN gene. The reverse primer 5 '-CCATGTAATTGAATCCCCCAACACTAGC-3', (SEQ ID NO: 17) extends the HPIV3 numbers 11448-11475, located in the L gene. The template for PCR was p3 / 7 (131) 2G-Stu. The PCR fragment 2 that resulted from this reaction contains at least 35 nt of the measles HA ORF and about 2800 nt of the ORF L of PIV3 and is flanked by an Xmal site and a Sphl site (occurring naturally at position 11317 of HPIV3). The third PCR reaction amplified the major central portion of the measles ORF HA from the cDNA template pTM-7, a plasmid containing the ORF HA of the Edmonston strain of measles virus supplied by the ATCC. Sequence analysis of this plasmid showed that the ORF HA of the measles virus contained in PTM-7 contains 2 pTM-7 amino acid differences of the Edmonston wild-type HA sequence used for insertion into the NP and MP junction and these were in the positions 46 of the amino acid (F to S) and in position 481 (Y to N). The direct primer 5'-CGGATAAACGCG2 CTACAAAGATAACC-3 '(SEQ ID No. 18) (MluI site in italics) and the reverse primer 5'-CGGATAAACGCGTTCTACAAAGATAACC-3 * (SEQ ID NO. 18) (' Xmal site in italics) amplified PCR fragment 3 containing nts 19-1838 of the ORF HA of measles. To assemble the pieces, PCR fragment 1 was digested with Stul and MluI while PCR fragment 3 was digested with MluI and Xmal. These two deferred fragments were then cloned by triple ligation in the Stul-Xmal window of pUC118 which had been modified to include a Stul site in its multiple cloning region. The resulting plasmid was digested with pUC118 (HA 1 + 3) while the Stul and Xmal fragment while the PCR fragment 2 was digested with Xmal and Spiil. The two digested products were then cloned into the Stul-Sphl window of p3 / 7 (131) 2G-Stu, resulting in plasmid PFLC (HA HN-L). The Stul-Spñl fragment, which includes the complete measles HA ORF, was then sequenced using reaction ready for Rhodaminad Terminator Cycle Sequences (ABI prism, PE Applied Biosystems, Foster City, CA). The chimeric construction sequence was confirmed. In this way, the ORF HA of the measles virus flanked by the HPIV3 transcription signals was inserted as an extra gene at the N / P, P / M, or HN / L junction of an antigenomic cDNA vector comprising an HPIV3 type wild or in the union N / P. or P / M of an antigenomic cDNA vector comprising an attenuated HPIV3.

Recovery of wild-type chimeric PIV3r and cp45Lr expressing the measles virus HA protein The five full-length vector cDNAs carrying the measles ORF HA as a separate gene were transfected separately into HEp-2 cells in six six-well plates cavities (Costar, Cambridge, MA) together with the support plasmids. { pTM (N), PTM (P no C), and pTM (L)} and LipofectACE (Life Technologies), and the cells were simultaneously infected with MVA-T7, a replication-defective vaccinia virus recombinant encoding the polymerase protein of the T7 bateriophage as previously described (Durbin et al., Virology 235: 323-332, 1997; Durbin et al., Virology 234: 74-83, 1997, each incorporated herein by reference). pTM (P no C) is a derivative of pTM (P) (Durbin et al., Viroloqy 261: 319-330, 1999) in which the expression ORF C had been silenced by the mutation of the start codon C. After incubation at 32 ° C for three days, the transfection collection was passed over a fresh monolayer of Vero cells in a T25 flask and incubated for 5 days at 32 ° C (referred to as step 1). The presence of HPIV3 in the collection of step 1 was determined by plaque titration on LLC-MK2 monolayer cultures with plaques visualized by immunoperoxidase staining with HPV3-specific HN-specific monoclonal antibodies and HA-spec measles lobes as previously described (Durbin et al., Virology 235: 323-332, 1997, incorporated herein by reference). The PIV3r virus (HA HN-L) present in the supernatant of the appropriate step 1 harvest was biologically cloned by plaque purification three times on LLC-MK2 cells as previously described (Hall et al., Virus Res. 22: 173 -184, 1992, incorporated herein by reference).

PIV3r (HA NP), cp45Lr (HA NP), PIV3r (HA PM) and cp45Lr (HA PM) were cloned biologically from their respective step 1 collections by terminal dilution using double serial dilutions in 96-well plates (12 cavities by dilution) of Vero cell monolayers. Biologically cloned recombinant viruses from the third round of plaque purification or the second or third round of terminal dilution were then amplified twice in LLC-MK2 cells. { PIV3r (HA HN-L.) Or Vero cells { PIV3r (HA N-P), cp45Lr (HA N-P), PIV3r (HA P-M), cp45Lr { HA P-M)} at 32 ° C to produce virus for further characterization. As in the first step, in the confirmation and characterization of the recombinant chimeric PIV3 that express the measles virus HA glycoprotein, each collection of step 1 was analyzed by RT-PCR using three different pairs of primers; a pair of each location of the ORF HA insert. The first primer pair amplified a PIV3 fragment extending nucleotides 1596-1968 of the full-length HPIV3 genome, which includes the N / P insertion site. This fragment size was increased to 2298 nucleotides with the measles ORF HA inserted between the N and P genes. The second primer pair amplified a PIV3 fragment extending to nucleotides 3438-3866 of the full-length HPIV3 genome, which includes the insertion site P / M. With the measles ORF HA inserted between the P and M genes, this fragment size increased to 2352 nucleotides. The third primer pair amplified a fragment of PIV3 which extends nucleotides 8466-8649 of the full-length antigenome. With the measles HA ORF inserted between the HN and L genes, this fragment size increased to 2211 nucleotides, which includes the HN / L insertion site. The five recovered viruses contained an insert of appropriate size in the appropriate location. The generation of each PCR product depends on the inclusion of reverse transcriptase, which indicates that each was derived from RNA and not from contaminating cDNA. Monolayers of LLC-MK2 cells were infected in T25 flasks in a multiplicity of infection (MOI, for its acronym in English) of 5 with any cp45Lr (HA N-P), cp45Lr (HA P-M), JSr or were infected for testing. The Vero cell monolayers in the T25 flasks were infected with the wild-type Edmonston strain of measles virus at an MOI of 5. Vero cell monolayers were selected for measles Edmonston virus infection because the measles virus It did not grow well in LLC-MK2 cells. At 24 hours after infection, the monolayers were washed with methionine-minus DMEM (Life Technologies). 35S methionine was added to the DMEM-minus medium at a concentration of 10uCi / ml and 1 ml was added to each flask which was then incubated at 32 ° C for 6 hours. The cells were harvested and washed 3 times in PBS. The cell granules were resuspended in 1 ml of RIPA buffer. { 1% (w / v) sodium deoxycholate, 1% (v / v) Triton X-100 (Sigma), 0.2% (w / v) SDS, 150mM NaCl, 50mM Tris-HCl, pH 7.4} , frozen-liquefied and clarified by centrifugation at 6500 XG for 5 minutes. The cell extract was transferred to an eppendorf tube and a monoclonal antibody mixture recognizing the measles virus HA glycoprotein (79-XV-V17, 80-III-B2, 81-1-366) was added (Hummel et al. J. Virol 69; 1913-6, 1995; Sheshberadaran et al., Ch. Virol. 83: 251-68, 1985, each incorporated herein by reference) or recognizing the HN protein (10 I / I, 403/7, 166/1 1) of PIV3 (van Wyke Coelingh et al., Virology 160 : 465-72, 1987, incorporated herein by reference) to each sample and incubated with constant mixing for 2 hours at 4 ° C. Immune complexes were precipitated by adding 200 μ? of a 10% suspension of protein A Sepharose beads (Sigma, St. Louis, MO) to each sample followed by constant mixing at 4 ° C overnight. Each sample was suspended in 90 μ? of shock absorber IX and 10 μ? of reducing agent. After heating at 70 ° C for 10 minutes, 20 μ? of each sample were loaded on a 4-12% polyacrylamide gel (NuPAGE, Novex, San Diego, CA) per the manufacturer's recommendations. The gel was dried and autoradiographed (Figure 2). The cp45Lr (HA P-M) and cp45Lr (HA N-P) coded for a protein precipitated by anti-measles HA monoclonal antibodies that was the same size as the authentic measles HA protein. The cp45Lr (HA PM) and cp45Lr (HA NP) expressed the measles virus HA protein to a greater degree than the wild-type Edmonston strain of measles virus which indicates that these constructs efficiently expressed the HA of the measles virus of the junctions N / P and P / M of the attenuated cp45Lr strain. The cp45Lr (HA N-P) and cp45Lr (HA P-M) were confirmed to be based on HPIV3 for their reactivity with the anti-HN monoclonal antibodies of PIV3.

The temperature sensitivity of the replication of precursor virus PIV3r and chimeric PIV3r (HA) in vitro The level of sensitivity to temperature of the replication of the chimeric PIV3r carrying the measles virus insertion was evaluated to estimate whether the acquisition of the HA insert modified the level of replication in the chimeric virus compared to the precursor vector virus at various temperatures (Table 1). Serial 10-fold dilutions of cp45Lr, cp45Lr (NP), cp45Lr (HA PM), PIV3r (HA HN-L), PIV3r (HA PM) or JSr in L-15 supplemented with 5% FBS were carried out. 4 mM glutamine and 50 g ml of gentamicin in LLC-MK2 cell monolayers in 96-well plates and incubated at 32, 36, 37, 38, 39, or 40 ° C for 6 days. The virus was detected by hemadsorption and reported as logioTCIDso / ml. Interestingly, the chimeric derivatives of the two wild-type vector viruses carrying the HA gene of measles virus, PIV3r (HA NN-L) and PIV3r (HA PM), were slightly restricted in replication at 40 ° C (Table 1) . The two attenuated PIV3r carrying the HA gene of the measles virus, cp45Lr (NP) and cp45Lr (HA PM), had a level of sensitivity to temperature similar to that of the vector virus, precursor of cp45Lr with cp45Lr (HA PM) which is more lightly ts than its predecessor. In this way, the viruses carrying the replicated inserts in the tissue culture similarly to the PIV3r precursor vector from which they were derived, with only a slight increase in sensitivity in temperature. These results indicate that PIV3r can easily serve as a vector to suit the HA insert at different sites without a major alteration in in vitro replication, and that PIV3r (HA) viruses can easily adapt to the addition of one or more mutations mitigating Table 1 - Replication at tolerant and elevated temperatures of recombinant HPIV3 expressing the measles virus HA protein as an extra gene at the N-P, P-M, or HN-L junctions. Virus titre (log10TCID50 / ml) at the indicated temperature Virus 32 ° C1 36 • c 37eC 38"C 39eC 40eC cp45Lr2 B .2 8. .2 7.2 5.26 3. .4 3.0 cp45Lr (HA PM) 3 7.4 6. 7 5.2 4.2 1. .4 1.4 cp45L (HA NP) 3 7.4 7. 2 5.7 4.2 2. .2 = 1.2 PIV3r (HA HN-L) 4 7.7 8. 2 7.0 7.7 6. .7 5.2 PIV3r (HA PM) 4 7.7 7. 4 6.7 6.2 6. .2 4.7 PIV3-JSr5 8.7 9. .0 9.0 8.4 8. .2 9.0 Tolerant temperature. Derivative ts recombinant of wild type strain JS of HPIV3, which carries 3 substitutions of attenuating amino acid derived from cp45. 3. Derivative ts attenuated recombinant wild type HPIV3 JS expressing the HA protein of the measles virus. 4. HPIV3 recombinant wild type expressing the HA protein of the measles virus. 5. JS strain of recombinant wild-type HPIV3. 6. The underlined title represents the lowest restrictive temperature at which a reduction of 100 times or greater is observed in the titer of that at 32eC and defines the virus deactivation temperature.

EXAMPLE II Chimeric PIV3r carrying an antigenic determinant of measles virus replicates effectively in hamster and induces high titers of antibodies against both HPIV3 and measles Determination of the level of replication and immunogenicity of PIV3r (HA) viruses in hamsters The level of replication of the chimeric PIV3r carrying an antigenic determinant of the measles virus was compared with that of its precursor PIV3r to determine whether the acquisition of the determinant, exemplified by an HA insert, significantly modified its ability to replicate and to induce a response immune in vivo. In two different experiments, groups of 6 or 7 Golden Syrian hamsters from 4 to 6 weeks of age were inoculated intranasally with 0.1 ml of EMEM (Life Technologies) containing 106 · 0 PFU of JSr, cp45Lr, cp45Lr (HA PM), cp45Lr (HA NP), PIV3r (HA HN-L), or PIV3r (HA PM) (Tables 2 and 3). On day 4 after inoculation, the hamsters were sacrificed and the nasal lungs and turbinates were harvested. The nasal turbinates and lungs were homogenized in a suspension at 10% or 20% of L-15 (Quality Biologicals, Gaithersburg, MD) respectively, and the samples were quickly frozen. The virus present in the samples was titrated in 96-well plates of LLC-MK2 cellular monolayers and incubated at 32 ° C for 7 days. The virus was detected by haemadsorption and the average logi0TCID5o / g was calculated for each hamster group. The insertion of the HA gene in wild type JSr (Table 2) restricted its replication from 4 to 20 times in the upper respiratory tract and up to five times in the lower respiratory tract, indicating only a slight effect of the acquisition of the HA gene in the replication of wild type JSr virus in hamsters. The replication of each of the two antigenic chimeras rcp45 (HA) was 10 times lower in the upper respiratory tract of hamsters (Table 3) than of cp45Lr, the recombinant precursor virus carrying the three attenuating ts mutations in the L protein, although it was the same as the precursor cp45Lr in the lower respiratory tract. Thus, for each of the two antigenic chimeras rcp45 (HA) there was a slight but statistically significant reduction in the replication in the upper respiratory tract of the hamsters, which indicates that the acquisition of the HA gene by cp45Lr increased its attenuation for the upper respiratory tract but not for the lower one. In this way, the effect of the insertion of the HA gene on the replication of wild type or attenuated PIV3 was comparable in the upper respiratory tract.

Table 2: Replication of wild-type chimeric virus PIV3r (HA) in the upper and lower respiratory tract of hamsters Virus title (log10TCID50'gm ± S. E .2) Virus1 # of [Tukey-Kramer clustering] 3 animals | Turbines nasal lungs cp45Lr 8 4., 0 ± 0.1 [A] 1.5 ± 0 .1 [A] PIV3r (HA N-P) 8 5,, 1 ± 0.1 [B] 5.9 ± 0 • 1 [B] PIV3r < HA P-M) 8 5. .9 ± 0.1 [C] 6.7 ± 0 • 2 [C] PIV3r (HA HN-L) 8 5., 9 ± 0.2 [C] 5.8 ± 0. ltB] JSr 8 6., 5 ± 0.1 [D] 6.6 + 0 • 2 [C] 1. The animals received 106 TCID5 ° of the indicated virus administered intranasally in an inoculum of 0.1 ml and the lungs and nasal turbinates were harvested 4 days later. Standard error The means of the virus titers were assigned for statistically similar groups (A-D) by the Tukey-Kramer test. Therefore, it means that each of the columns with different letters are significantly different (= 0.05) and those with the same letter are not significantly different.

Table 3: Replication of the antigenic chimeric viruses PlV3rcp45L (HA) in the upper and lower respiratory tract of hamsters Virus title (log10TCID50 / gm ± S. E.2) Virus1 # of [Tukey-Kramer] 3 animals Turbinates nasals Lungs cp45Lr 7 ± 0.2 [A] 9 ± 0.1 [A] cp45Lr (HA NP) 7 ± 0.2 [B] 9 ± 0.1 [A] cp45Lr (HA PM) 7 ± 0.1 [B] 9 ± 0.2 [A] JSr 5 ± 0.1 [C] 6 ± 0.2 [B] The animals received 10 pfu of the indicated virus administered intranasally in an inoculum of 0.1 ml and the lungs and nasal turbinates were harvested 4 days later. Standard error The means of the virus titers were assigned to statistically similar groups (A-D) by the Tukey-Kramer test. Therefore, it means that each of the columns with different letters are significantly different (< x = 0.05) and those with the same letter are not significantly different.

The ability of chimeric HPIV3r (HA) viruses to induce an immune response for HPIV3 and for the measles virus is discussed below. Groups of 6 to 24 Golden Syrian hamsters (4 to 6 weeks old) were infected as described above with either 106-0 PFr JSr, PIV3r (HA PM), cp45Lr, cp45Lr (HA PM), or cp45Lr (HA NP) (Table 4) on day 0. The serum was collected from each hamster on day 1 and on day 25 after inoculation. The serum antibody response to HPIV3 was evaluated by haemagglutination-inhibition analysis (HAI) as described above (van Wyke Coeling et al., Virology 143; 569-582, 1985, incorporated herein by reference). reference), and the serum antibody response for measles virus was evaluated by 60% plaque reduction assay as described previously (Coates et al., Am. J. Epidemiol., 83: 299-313, 1966, incorporated in the present as a reference). These results were compared with those of the additional control group of cotton rats that received 105-0 of attenuated measles virus in vivo (Moraten strain) administered intramuscularly on day 0. Cotton rats, instead of hamsters, were used. in this group because the measles virus is infectious only weekly for hamsters. As can be seen in Table 4, each of the PIV3 (HA) chimeric viruses was capable of producing an important serum neutralizing antibody response against the measles virus. There was not a significant difference between the amount of serum neutralizing antibody produced by the attenuated derivative of cp45Lr (HA P-M) as compared to its counterpart in PIV3r (HA P-M), wild-type background. In addition, the level of measles virus-neutralizing serum antibodies induced by the PIV3r (HA) recombinants were on average 5 times higher than those achieved by intramuscular immunization with the attenuated measles virus vaccine in vivo. In addition, the serum antibody response to HPIV3 produced by all chimeric viruses was also important and comparable to that produced by infection with wild type JSr.

Table 4. Antigenic chimeric viruses PIV3r (HA) that produce an excellent serum antibody response for both measles virus and PIV3 Virus1 Response Title Serum antibody serum antibody to HPIV3 virus measles animals (titer (titrant a HAI, mean reciprocal neutralization reduction of log2 ± SE) plate at 60%, mean reciprocal logz ± SE2) Day 0 Day 25 Day 0 Day 25 cp45Lr3 18 < 3. .3 ± 0 < 3 .3 ± 0 = 2, .0 ± 0 10.7 ± 0.2 cp45Lr (HA P-M) 4 24 = 3. .3 ± 0 12. 8 ± 0. 1 < 2,, 0 ± 0 9.2 ± 0.2 cp45Lr (HA N-P) 5 6 = 3. , 3 ± 0 13. 4 ± 0. 4 < 2. .0 ± 0 10.8 ± 0.3 PIV3r (HA P-M) 6 6 = 3, .3 ± 0 13. 3 ± 0. 3 = 2 .0 ± 0 10.3 ± 0.2 Measles virus (Móraten) 7 4 = 3. , 3 ± 0 10. 8 ± 0. 2 < 2., 0 ± 0 = 2. O ± 0 JSrB 6 = 3. .3 ± 0 = 3 .3 ± 0 < 2., 0 ± 0 10.7 ± 0.2 The virus was administered at a dose of 106'0 PFU in an inoculum of 0.1 intranasally on day 0 to all animals except those in the group of measles virus that received the virus by intramuscular injection. Standard HPIV3 attenuated standard error with three temperature sensitive mutations (ts) in the L protein, derived from cp45. Recombinant attenuated HPIV3 in the cp45L background with the O F HA of the measles virus in the P / M non-coding region of PIV3r. Recombinant attenuated HPIV3 in the cp45L background with the ORF HA of measles virus in the non-coding region N / P of PIV3r.

Recombinant HPIV3 with the 0RF HA of the measles virus in the non-coding region P / M of the wild-type PIV3r.

Live attenuated measles vaccine virus, strain Moraten, was administered at a dose of 105 pfu in an inoculum of 0.1 by intramuscular injection to 4 rats of cotton in a separate study. All the other animals were hamsters. HPIV3 recombinant wild type.

Six hamsters from each group and from a control group similarly infected with RSV were inoculated on day 25 with 106"0 pfu of HPIV3 wild type biologically administered intranasally administered virus in an inoculum of 0.1 ml.The lungs and nasal turbinates were collected. on day 4 and processed as described above, the virus present in the samples was titered in 96-well plates of LLC-MK2 cell monolayers and incubated at 32 ° C for 7 days.The virus was detected by haemadsorption and the mean logioTCID5o / g was calculated for each group of hamsters.As shown in Table 5, those hamsters that had received the chimeric viruses, either in the attenuated or wild-type structure were fairly protected against replication of wild-type HPIV3 inoculation both in the upper and lower respiratory tract, in this way, despite the slight attenuation effect of the acquisition of the HA gene of the virus d In the replication of the cp45r (HA) chimeric viruses, infection with either cp45Lr (HA PM) or cp45Lr (HA NP) induced a high level of protection against HPIV3 as indicated by approximately a 1000-fold reduction in its replication in the upper and lower respiratory tract of hamsters. Because the wild-type measles virus does not replicate efficiently in hamsters, it was not used to inoculate this host. However, it is expected that candidates for attenuated chimeric cp45Lr (HA) vaccine will be quite effective against measles virus because high levels of neutralizing antibody were induced, ie a mean titer of more than 1: 5000. Comparable levels of measles virus antibodies were associated with strong resistance to disease by the measles virus in humans (Chen et al., J. Infect. Dis. 162: 1036-42, 1990, incorporated herein by reference). ).

Table 5. HA chimeric HPIV3 measles chimeric and wild type chimeric viruses that are highly protective against replication of the wild type PIV3 inoculated in the upper and lower respiratory tracts of hamsters Virus title Reduction in (log10TCID50 / g) [Aggregate (logi0) pamiento Tukey-Kramer3] Animals # Turbinados Turbinados immunized with nasal animals Nose lungs Lungs RSV 6 7.0 ± 0.3 [A] 5.7 ± 0.4 [A] NA2 NA cp45Lr (HA PM) 6 3.4 ± 0.3 [B] 2.9 ± 0.0 [B] 3.6 2.8 qp 5L (HA NP) 6 2.6 ± 0.3 [B] 3.4 ± 0.2 [B] 4.4 2.3 PIV3r (HA P-M) 6 2.0 ± 0.3 [B] 3.2 ± 0.1 [B] 5.0 2.5 cp45Lr 6 1.9 ± 0.2 [B, C] 3.6 ± 0.1 [B] 5.1 2.1 JSr 6 < 1.4 ± 0.0 [C] 2.9 ± 0.2 [B] > 5.7 2.8 All groups were inoculated with 106 pfu of wild-type PIV3 biologically derived in an inoculum of 0.1 ml administered intranasally. Not applicable. The means of virus titers were assigned to statistically similar groups (A-C) by the Tukey-Kramer test. Therefore, it means that each of the columns with different letters are significantly different (a = 0.05) and those with the same letter are not significantly different.

EXAMPLE III Construction of the antigenomic cDNAs encoding a chimeric HPIV3-1 vector carrying an HPIV2 HN gene as an extra transcription / translation unit inserted between the F and HN genes and the recovery of infectious viruses PIV3r-l is a recombinant chimeric HPIV3 in which the HN and F genes have been replaced by those of HPIV1 (see, for example, Skiadopoulos et al., Vaccine 18: 503-510, 1999; Tao et al., Vaccine 12: 1100-1108 , 1999, U.S. Patent Application Serial No. 09 / 083,793, filed May 22, 1998; U.S. Patent Application Serial No. 09 / 458,813, filed December 10, 1999; of U.S. Patent No. Serial No. 09 / 459,062, filed December 10, 1999, each incorporated herein by reference). In the present example, the HN gene of HPIV2 was inserted into the PIV3r-1 virus that served as a vector to produce a chimeric derived virus, which carries a heterologous antigenic determinant introduced from HPIV2, capable of protecting both HPIV1 and HPIV2. The HN gene of HPIV2 was also inserted into an attenuated derivative of PIV3r-1, designated PIV3r-Icp45, which contains 12 of the 15 cp45 mutations, ie those mutations in genes other than HN and F, inserted in the PIV3r structure (Skiadopoulos et al., Vaccine 1_8: 503-510, 1999). The source of wild type virus HPIV2 was the wild-type strain V9412-6 (designated PIV2 / V94) (Tao et al., Vaccine 17: 1100-1108, 1999), which was isolated in Vero cells from a nasal wash that It was obtained in 1994 from a child with a natural HPIV2 infection. PIV2 / V94 was a plaque purified 3 times on Vero cells before being amplified twice in Vero cells using OptiMEM tissue culture medium without FBS. A cDNA clone of the PIV2 / V94 HN gene was generated from virion RNA by reverse transcription (RT) using random hexamers and Superscript Preamplification System (Life Technologies) followed by PCR using an Advantage cDNA Synthesis kit (Clontech, Palo Alto, CA) and synthetic primers that introduced the Ncol-Hindl II sites flanking the HN cDNA (Figure 3A). The sequences of these primers were: (with HPIV specific sequences in uppercase, underlined restriction sites, nts that are without HPIV or that were altered from wild-type in lowercase letters and initial codons and ending in bold), HN from HPIV2 in the 5 'direction -gggccATGGAAGATTACAGCAAT- 3 '(SEQ ID NO.19); HN of HPIV2 in the 3 ', 5' -caataagcTTAAAGCATTAGTTCCC-3 '(SEQ ID NO 20). PCR fragments of HN were digested with NcoI-HindIII and cloned into pLit. PIV31HNhc to generate pLit.32HNhc (Figure 3B). The heterologous HN gene insert of HPIV2 in pLit.32HNhc was completely sequenced using the ThermoSequenase kit and the 33P-tagged terminators (Pharmacia Amershaiti, Piscataway, NJ) and was confirmed to contain the authentic sequence of the HN coding region of PIV2 / 94 . The HN gene of HPIV2 in pLit.32HNhc was further modified by PCR and Deep Vent thermostable DNA polymerase (New England Biolab, Beverly, MA) to introduce the PpuMI sites for cloning into the single PpuMI site in p38 'APIV31hc, Figure 3C ( Skiadopoulos et al., Vaccine 18: 503-510, 1999). The sequences of these primers were (with HPIV specific sequences in upper case, relevant restriction sites underlined, nt without HPIV or altered nt from low wild type): HN of HPIV2 in the 5 'direction, 5'-gcgatgggcccGAGGAAGGACCCAATAGACA-3' ( SEQ ID No. 21); HN of HPIV2 in the 3 'direction, 5'-cccgggtcctgATTTCCCGAGCACGCTTTG-3' (SEQ ID NO.22). The modified cDNA carrying the HPIV2 HN ORF consists of (from left to right) a partial 5 'untranslated region (5'-UTR) of HN from HPIV3 that includes the PpuMI site at the 5' end, the HN ORF of HPIV2, the HN UTR 3 of HPIV3, a complete set of HPIV3 transcription signals (ie, gene end sequences, intergenic region and gene start) whose sequences are compatible in the binding of HIV and L gene of HPIV3, a partial 5 'UTR of HPIV3 L and a PpuMI site added at its 3' end (Figure 3C). This fragment was digested with PpuMI and inserted into p38'APIV31hc digested with PpuMI to generate p38 * APIV31hc .2HN (Figure 3D). The inserted PpuMI cassette was sequenced in its entirety and found to be as it was designated. The p38 'APIV31hc .2HN insert was isolated as an 8.5 kb BspEI-Sphl fragment and inserted into the BspEI-Sphl window of pFLC.2G + .hc or pFLCcp45 to generate pFLC.31hc.2HN or pFLC.31hc.cp45.2HN , respectively (Figure 3, E and F). pFLC.2G + .hc and pFLCcp45 are full-length antigensic clones that encode PIV3r-ly and PIV3rcp45 / respectively, as described previously (Skiadopoulos et al., J. Virol 7: 1374-81, 1999; Tao et al. , J. Virol. 72: 2955-2961, 1998, each is incorporated herein by reference). Confluent HEp-2 cells were transfected with pFLC.31hc.2HN or pFLC.31hc. cp45.2HN plus pTM (N), PTM (P no C) and PTM (L) support plasmids in the presence of MVA-T7 as described previously (Durbin et al., Virology 235: 323-332, 1997, incorporated in the present as a reference). The recombinant chimeric viruses recovered from the transfection were activated by the addition of TPCK trypsin (Catalog No. 3741, Worthington Biochemical Corp., Freehold, NJ) as all were passages and virus titers carrying the HPV1 HN and F glycoproteins. as described above (Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference). The recovered chimeric recombinant viruses PIV3r-1.2HN and PIV3r-lcp45.2HN were purified by plate-to-plate passage in monolayer LLCMK2 in agarose coat as described above (Tao et al., Vaccine 17: 1 1 001 108, 1999 , incorporated herein by reference). To determine whether the recombinants PIV3r-1.2HN and PIV3r-lcp45.2HN contain the HN gene of HPIV2 heterologous, the viral RNA of each recovered recombinant chimeric virus was amplified in LLC-K2 cells and concentrated by precipitation of polyethylene glycol (PEG, its acronym in English) (Mbiguino et al., J. Virol. Methods 31: 161-170, 1991, incorporated herein by reference). The virion RNA (vRNA) was extracted with Trizol (Life Technologies) and used as a template to synthesize the first strand cDNA using the Superscript Preamplification system (Life Technologies, Gaithersburg, MD) and random hexamer primers as described above. The synthesized cDNA was amplified by PCR with the Advantage cDNA synthesis kit (Clontech, Palo Alto, CA) with primers specific for the F coding region of HPIV1 and HN of HPIV1 (for F of HPIV1 5'-AGTGGCTAATTGCATTGCATCCACAT-3 '(SEQ ID No. 23) and for HPIV1 HN 5 '-GCCGTCTGCATGGTGAATAGCAAT-3') (SEQ ID NO.24). The relative locations of primers F and HN of PIV1 are indicated by the arrows in Figures 3 and 4. The amplified DNA fragments were digested and analyzed on agarose gels (Figure 4). Data for PIV3r-1 cp45.2HN are not shown, although they were comparable and confirmed in structure. PIV3rl.2HN and PIV3r-lcp45.2HN each contained the insert of the expected size and digestion patterns with a number of restriction enzymes confirmed the identity and authenticity of the inserts. The presence of cp45 mutations in PIV3r-lcp45.2HN was also confirmed. To confirm HN expression of HPIV2 by the chimeric virus PIV3r-1.2HN, LLC-MK2 monolayers in T25 flasks were infected with PIV2 / V94, PIV3r-l or PIV3r 1.2HN at an MOI of 5 in 5 ml of serum-free OptiMEM containing 0.5 g ml of TPCK trypsin. After incubation for 18 hours at 32 ° C, the flasks were washed three times with .5 ml of methionine and deficient DMEM of cysteine (Bio Whittacker, Walkersville, MD). The cells were then fed with 1 ml of methionine-deficient DMEM and cysteine supplemented with 120 μ? of a mixture of ProMix 35S-methionine and 35S-cysteine (Pharmacia Amersham, Piscataway, NJ) and incubated for 18 hours at 32 ° C. The cells were scraped into the medium, pelleted by brief centrifugation in a microcentrifuge and washed three times with cold PBS. Each cell pellet was resuspended in 1 ml of RIPA buffer (1% sodium deoxycholate, 1% Triton X-100, 0.2% SDS, 150 mM NaCl and 50 nM Tris-HCl, pH 7.4) containing 250 units / ml of Benzonase (Sigma), frozen / liquefied once and clarified by centrifugation at 12,000Xg for 5 minutes in a microcentrifuge. The clarified supernatants were transferred to a clean microcentri leak tube, mixed with 50 μ? of monoclonal antibody HN antiHPIV2 (Abm) 150S1 (Tsurudome et al., Virology 171: 38-48, 1989, incorporated herein by reference) and incubated with mixing at 4 ° C for 3 hours. The monoclonal antibody was precipitated by adding to each tube 0.2 ml of 10% protein A sepharose suspension (in RIPA buffer) and incubation with mixing at 4 ° for 18 hours. The beads were washed three times with RIPA buffer and pelleted by brief centrifugation in a microcentri leak Each sample was. suspended in 90 μ? of shock absorber IX and 10 μ? was resolved on a 4-12% SDS polyacrylamide gel (PAGE NOVEX, San Diego, CA). The gel was dried and autoradiographed (Figure 5). The Abm, specific for HN of PIV2, precipitated a protein from LLC-K2 cells infected by both PIV3r-1.2HN and PIV2 / V94, but not from cells infected with PIV3r-l, with an expected size for the 86kD Kd HN protein of HPIV2 (Rydbeck et al., J. Gen. Virol. 69: 931-5, 1988, incorporated herein by reference).

EXAMPLE IV PlV3r-l viruses carrying an HPIV2 antigenic determinant exhibit temperature sensitive phenotypes similar to those of their precursor vector viruses The level of temperature sensitivity of replication of PIV3r-1.2HN and PIV3r-l.cp45.2HN in LLC-MK2 cells was evaluated to determine whether the acquisition of the ORF HN of HPIV2 by the wild type or attenuated viruses of PIV3r-l used as vectors altered the level of sensitivity to the temperature of the replication in the resulting chimeric derivatives that carry the Heterologous antigenic determinant of HPIV2 compared to vector precursor viruses (Table 6). PIV3r-1.2HN and PIV3r-lcp45, 2HN, along with control viruses, were serially diluted 1:10 in IX L15 supplemented with 0.5.μ? / P? 1 TPCK trypsin and used to infect monolayers LLC-MK2 in plates of 96 cavities in quadruplicate. The infected plates were placed at various temperatures for 7 days before virus titers were determined by haemadsorption using 0.2% guinea pig erythrocytes (in IX PBS). Virus titers were presented as log10TCID5o ± standard error (S.E., for its acronym in English). As shown in Table 6, PIV3r-1.2HN and PIV3r-Icp45.2HN exhibited a level of sensitivity to temperature similar to that of their precursor vector viruses, ie PIV3r-ly and PIV3r-lcp45 / respectively, each of which lacks of the HN insert of HPIV2. This indicated that the introduction of an extra transcription / translation unit in PIV3r-1.2HN and PIV3r-1 cp 5.2HN did not significantly alter the level of sensitivity to replication temperature in vitro.

Table 6. PIV3r-l viruses carrying the HN insert of PIV2 have a temperature sensitive phenotype similar to that of their precursor virus. Title at 32 ° Ca Title reduction (logioTCID50) to Virus (log10TCID50) various temperatures (cC) at 35o 36 ° 37 * 38 ° 39 ° 40 ° PIV2 / V9412 7. 8 Ó.3 (0. l) c 0.0 (0.4) (0.4) 0.0 PIV1 / Washing 64 8. 5 1.5 1 .1 1.4 0.6 0.5 0.9 PIV3r / JS 7. 9 0.3 0, .1 0.1 (0.3) (0.4) 0.4 PIV3 cp 5 7. 8 0.5 0, .3 1.3 3.4d 6.8 6.9 PIV3r-l 8. 0 0.8 0, .5 0.6 0.9 1.1 2.6 PIV3r-1.2HN 8. 3 0.5 (0, .3) 0.3 0.6 1.5 2.6 PIV3r-lcp45 8. 0 0.5 0, .4 3.4 4.8 6.6 7.5 PIV3r-lcp45. 2HN 8. 0 0.3 1, .4 2.9 5.3 7.6 7.6 The presented data are means of two experiments. The data at 35 ° C were from an individual experiment. The numbers in parentheses represent an increase in the title The underlined value indicates the deactivation temperature at which the virus titre showed a reduction of 100 times or more compared to the titre at 32 ° C.

EXAMPLE V Replication and immunogenicity of HPIV3r-1.2HN chimeric viruses in animals To determine the level of replication of chimeric viruses in vivo Golden Syrian hamsters in groups of six were inoculated intranasally with 0.1 ml of medium IX L-15 containing 105- 3TCID50 (or 106 pfu) of virus (Table 7). Four days after infection, the hamsters were sacrificed and their nasal lungs and turbinates collected. The virus titers were determined, expressed as the logi0TCID5o average / gram of tissue (Table 7). PIV3r-1 expressing the HN gene of PIV2, designated PIV2r-1.2HN, is more restricted in replication than its precursor PIV3r-l as indicated by a 30-fold reduction in virus titer in the upper respiratory tracts as inferior of the hamsters. In this way, the insertion of the transcription / translation unit expressing the HN protein of PIV2 in PIV3-l attenuates the virus for hamsters. The attenuation effect of the insertion of a transcription / translation unit containing HF ORF of PIV2 in PIV3-l was slightly higher than that observed for the insertion of a similar unit containing the measles ORF HA in the recombinant JS strain of the PIV3 wild type. The virus PIV3r-1 cp 5.2HN was 1, 000 times more restricted in replication than the PIV3r-lcp45 precursor, which indicates that the attenuation effect of the HN insertion of PIV2 and the cp45 mutations are additive. It should be possible to adjust the level of attenuation as necessary by adding fewer than 12 cp45 mutations that are present in PIV3r-l.cp45.2HN.

Table 7. The chimeric PIV3r-l expressing the HN glycoprotein of PIV2 (PIV3r-1 .2HN) is attenuated in the respiratory tract of the hamsters Tissue virus titre Experiment No. Virus indicated (log10TCID5o / g ± S. E.) ° NT Lungs PIV3r-l 6. 9 ± 0. l [A] d 6.0 ± 0.3 [A] Ia PIV3r-1.2HN 5. 4 ± 0. 2 [B] 4.4 ± 0.4 [C] PIV3r-l 6. 7 ± 0. 1 [A] 6.6 ± 0.2 [A] PIV3r-l .2HN 5. 1 ± 0. 1 [B, C] 5.2 ± 0.2 [B] 2b PIV3r-lcp45 4. 6 ± 0. 3 [C] 1.8 ± 0.4 [D] PIV3r-lcp45.2HN 1. 5 ± 0. 1 [D] = 1.2 [D] PIV3r / JS 6. 5 ± 0. 2 [A] 6.7 ± 0.1 [A] rc 45 4. 9 ± 0. 2 [B, C] 1.2 ± 0.04 [D] a Groups of six animals were inoculated inthanasely with 106 pfu of the indicated virus in a 0.1 ml medium on day 0. b Groups of 6 hamsters were inoculated intranasally as in experiment 1 with 105'3 TCIDso of the virus indicated on day 0. c The lungs and nasal turbinates of the hamsters were harvested on day 4. The virus titers in the tissue were determined and the titer expressed as logioTCID50 / gram ± standard error (SE) NT = nasal turbinates. d The means in each column with a different letter are significantly different (a = 0.05) by the Duncan Multiple Interval test while those with the same letter are not significantly different.

Because the simple PIV3r-1.2HN virus expresses PIV1 protective antigens (glycoprotein F and HN) and PIV2 (the unique HN glycoprotein), infection with this virus will induce resistance against inoculation with wild type viruses either PIV1 or PIV2. To verify this, Golden Syrian hamsters in groups of 12 were immunized intranasally with 105'3 TCIDso of virus as described above. Half of the hamsters were inoculated with PIV2 on day 29. The remaining half with PIV1 on day 32. Lungs were collected from the hamsters and nasal turbinate tissues 4 days after inoculation and the virus titers inoculated were determined as described above (Table 8). Sera were obtained before and 28 days after the immunization and tested for their neutralizing antibody titer against PIV1 and PIV2.

Table 8. Chimeric virus PIV3r-l expressing the HN glycoprotein of PIV2 (PIV3r-1 .2HN) protects hamsters against inoculation with both PIV1 and PIV2 Virus Title of neutralizing antibody Title of virus inoculated in serum immunizer 3 against the indicated virus indicated tissues (mean reciprocal log2 ± SE) b (logioTCID50 / g ± SE) c PIVl PIV2 PIVl PIV2 pre post pre post NT lung NT lung PIV3r / JS = 4 .0 ± 0 .0 = 4 .0 ± 0 .0 4. 5 ± 0. 1 4 .6 ± 0.2 5 .4 ± 0. 2 5.1 ± 0.1 6 .8 ± 0. 2 6.0 ± 0.3 PIV2 = 4 .0 ± 0 .0 = 4 .0 ± 0 .0 4. 3 ± 0. 2 9 .6 ± 0.2 5 .7 ± 0. 2 5.7 ± 0.2 = 1.2 «S1.2 CVJ LO PIV3r-l 4. 2 ± 6. 1 8,, 5 ± 0. 3 4. 0 ± 0. 0 4 .2 ± 0.1 = 1.2 = 1.2 6 .3 ± 0. 1 6.5 ± 0.2 CM PIV3r- 1. HN = 4 .0 ± 0 .0 6,, 2 ± 0. 2 4. 1 ± 0. 1 8 .3 ± 0.2 2 .3 ± 0. 5 = 1.2 = 1.2 = 1.2 PIV3r-lcp45 = 4 .0 ± 0 .0 6 .2 ± 0. 4 = 4 .010 .0 4 .010.0 3 .6 ± 0. 3 2.710.5 6 .010. 1 5.7 ± 0.4 PIV3r-lcp45, 2HN 4. 0 ± 0. 9 4, .1 ± 0. 1 4. 0 ± 0. 0 4 .2 ± 0.1 5 .1 ± 0. 2 4.8 ± 0.2 6 .8 ± 0. 1 6.6 ± 0.2 Hamsters in groups of 12 were immunized with 10 TCID50 of the indicated virus intranasally on day 0. The serum was diluted 1:10 with OptiMEM and heat inactivated by incubation at 56 ° for 30 minutes. The serum neutralizing antibody titer was determined in LLC-MK2 and titers were expressed as the reciprocal mean log2 ± standard error (SE). Half of the hamsters in each immunized group were inoculated with 106TCID50PIV2 on day 29 and the remaining half was inoculated with 106TCID50PIV1 on day 32. Tissue samples were collected 4 days after inoculation and the inoculated virus titers were expressed as logi0TCID50 / gram of tissue ± SE. NT = nasal turbinates.

As expected, PIV3 did not provide resistance against either PIV1 or PIV2 (Tao, Vaccine 17: 1100-11 08, 1999), whereas previous infection with the wild type virus of PIV2 and PIV3r-l induced complete resistance to the virus. replication of the inoculated viruses PIV2 and PIV1, respectively - In contrast to these viruses that provided protection against only one virus, PIV3r-1.2HN induced antibodies for both PIV1 and PIV2 and included strong resistance for both PIV1 and PIV2 as indicated by the 1,000 to 10,000 fold reduction in the replication of each virus in the upper and lower respiratory tract of hamsters immunized against PIV3r-1.2HN. This indicated that a simple recombinant chimeric PIV can induce a residence against two human viral pathogens. However, the PIV3r-1.2HN derivative carrying the cp45 mutations failed to induce significant resistance to the replication of the wild type PIV1 or PIV2 inoculated virus indicating that this particular recombinant chimeric virus is over-attenuated in a hamster. The introduction of one or more selected mutations cp45, instead of the complete set of 12 mutations, in PIV3r-1.2HN can be performed to adjust the level of attenuation of PIV3r-1.2HN at an adequate level.

EXAMPLE VI Construction of the cDNAs encoding HPIV3r viruses containing nucleotide insertions As discussed above, the insertion of the measles ORF HA between the gene linkage either N / P or P / M of the attenuated vector virus, PIV3rcp45L, as well as in the N / P, P / M and HN / L junctions of wild-type PIV3, further restricted its replication in the upper respiratory tract of the hamsters, indicating that the insertion of an additional gene at any location within the HPIV3 genome can increase the attenuation of candidate vaccine viruses. In these exemplary aspects of the invention, the gene insert was relatively superior (approximately 1900 nts). Additional examples are provided herein that indicate that the size of the insert specifies a selectable level of attenuation of the resulting recombinant virus. This was evaluated by introducing sequences of various lengths that were derived from a heterologous virus, exemplified by the RSV strain A2, as single gene units (GUs) between the HN ORFs of HPIV3. The inserts were specifically designed so that they lack any significant ORF, so any observed effects might not be complicated by the possible contribution of expressed protein. In order to distinguish between the effects due to increased genome length against the expression of an additional A Nm, a second series of constructions was elaborated in which the inserts of similar sizes were introduced in the non-coding region in the 3f direction (NCR) of the HN gene. In this way, two series of PIV3r containing insertions of increasing length were made: in the Gü series, the insert was added as an extra gene that codes for an extra mRNA, while in the NCR series the insert was made from such form that the gene number will not change.

Construction of cDNAs encoding HPIV3r viruses containing 3 'GU and NCR insertions. Insertional mutations were constructed on a plasmid based on pUC, 118-Stu, which contains the Xhol fragment to Spñl (HPIV3 nts 7437-11317) of the full-length HPIV3 clone p3 / 7 (131) 2G-Stu. Two plasmids were constructed separately as acceptor plasmids for the insertion of the GUs and NCR 3 'extensions of the HN gene (Figure 6). In each, a synthetic duplex oligonucleotide containing multiple cloning sites was inserted into the unique Stul site. The sequence inserted for the GU insertion plasmid contained a gene end signal sequence (GE) of HN. The conserved intergenic trinucleotide (IG) sequence, and a geIi (GS) L start signal sequence, cis-acting sequences directing I, transcription termination and initiation of the HN gene of transcription of the inserted sequence, respectmente (Figure 6). Additional single restriction nuclease sites were included in the multiple cloning region to facilitate subsequent selection and subcloning. The acceptor plasmid of the 3 'NCR extension was designed and constructed similiarly, although it lacked the GE IG and GS cis-acting sequences at its 5' end (Figure 6B, Table 9). The d53RSV sites of RSV antigenomic plasmid or pUC 118FM2 subgenomic plasmid (Table 9) were digested with the appropriate restriction enzymes and fragments of the desired sizes were isolated by agarose gel electrophoresis and ligated individually at the single site to the NCR 3 'extension acceptor plasmid of the GU gene or the HN gene (Figure 6, Table 9). The clones were selected to identify ones in which RSV restriction fragments were inserted in the reverse orientation, an orientation in which all reading frames contained multiple terminator codons (Figure 7). Variation of synthetic short duplex oligonucleotides of 13 to 17 nucleotides were also inserted as necessary in the GU or the 3 'NCR acceptor plasmids to modify the length of the genome to conform to the "six rule" (Table 9). The specific RSV sequences and the size of the short synthetic oligonucleotides added are summarized in Table 9. The plasmid clones were sequenced through all the restriction enzyme sites used for subcloning, and the Xhol-Sphl fragments containing the insertion mutations conforming to the six rule, either as NCR extensions of the Gus gene or HN, were cloned into the full-length PIV cDNA plasmid p3 / 7 (l31) 2G +. An insert, containing the insert 1908 GU, was also placed in an antigenomic cDNA carrying the three L mutations of cp45.

Table 9: Nucleotide sources used to create the gene unit (GU) and insertions extension of non-coding region (NCR) 3 'of the HN gene Restriction Site Size and Insertion Site Insertion site fragment nt in the antigenome cloning GU (nts cloning NCR (nts restriction (nts) multiple RSV GU (58 multiple total NCR (32 total nt) + rule of 6 inserted) nt) + 6 inserted rule) oligonucleotidee oligonueleótidoe 97a Sspl-Sspl; 7272-7369 +58 +13 168 nd nd 212 Hpal-Hpal; 12243-12455 nd nd +32 +14 258 603 Sspl-Sspl; 309-912 +58 +17 678 nd nd 925b Hpal-Hpal; 12455-13380 +58 +13 996 +32 +15 972 1356b'c Hincll-Hincll; 5060-6417 +58 +14 1428 +32 +16 1404 1850b'd Hpal-Hpal; 12455-13380 +58 +0 1908 nd nd 3079b EcoRV-Ec / 13611; 1403- nd nd +32 +15 3126 4482 3845 Scal-Scal; 344-4189 +58 +15 3918 +32 +17 3894 a. The source of the RSV sequence is pUC118FM2, a plasmid containing a subgenomic cDNA fragment of subgroup A RSV as described above (Juhasz, K. et al, J Virol., 71: 5814-5819, 1997.) · b- The source of the RSV sequence is the D53 sites, a plasmid containing the complete RSV subgroup A cDNA sequence with various point mutations introduced as described above. The D53 sites of the plasmid described above were used to derive the rA sites of the virus described in Whitehead, S. et al. J. Virol., 72: 4467-4471, 1998. c. The 1356 nt fragment purified with gel contained a deletion of 1 nt compared to the predicted 1357 nt restriction of the restriction endonuclease cleavage product. d. The 1850 nt fragment is a two 3 'to 3' product bound to restriction fragments 925 nt. and. The following oligonucleotides were inserted into the MluI restriction site to form all of the above sequences inserted into the 6: 13mer rule: CGCGGCAGGCCTG (SEQ ID NO: 25); 14mer: CGCGGCGAGGCCTG (SEQ ID NO.26); 15mer: CGCGAGGCCTCCGCG (SEQ ID No. 27); 16mer: CGCGCCGCGGAGGCCT (SEQ ID No. 28); ] 7mer: CGCGCCCGCGGAGGCCT (SEQ ID NO.29). nd, not done.

Recovery of the recombinant PIV3 carrying the insertion mutants The full length antigenomic cDNA derivatives carrying the insertion mutations and the three support plasmids pTM (N), pTM (p no C) and pT (L) (Durbin et al. ., Virology 235: 323-332, 1997, Durbin et al., Virology 261: 319-330, 1999, each incorporated in a present as a reference) were transfected into HEp-2 monolayers in 6-well plates 6 (Costar, MA) using LipofectACE (Life Technologies, MD), and the monolayers were infected with MVA-T7 as described previously (Durbin et al., Virology 235: 323-332, 1997; Skiadopoulos et al., J. Virol. 72: 1762-8, 1998, each incorporated herein by reference). After incubation at 32 ° C for 4 days, the transfection collection was passed over LLC-2 cells in T-25 flasks which were incubated at 32 ° C for four to eight days. The supernatant of clarified medium was subjected to plaque purification in LLC-MK2 cells as described previously (Durbin et al., Virology 235: 323-332, 1997; Hall et al., Virus Res. 22: 173-184, 1992 Skiadopoulos et al., J. Virol. 72 ^: 1762-8, 1998, each incorporated herein by reference). Each biologically cloned recombinant virus was amplified twice in LLC-K2 cells at 32 ° C to produce the virus for further characterization. The virus was concentrated from the clarified medium by precipitation of polyethylene glycol (Mbiguino et al., J. Virol. Methods 31: 161-170, 1991, incorporated herein by reference), and the viral RNA (vRNA) was extracted with Trizol reagent (Life Technologies). Reverse transcription was performed on vRNA using the Superscript II Preamplification System (Life Technologies) with random hexamer primers. The Advafttage cDNA PCR kit (Clontech, CA) and the sense (PIV3 nt 7108-7137) and antisense primers (pIV3 nt 10605-10576) were used to amplify the fragments for restriction endonuclease digestion or sequence analysis. The PCR fragments were analyzed by agarose gel electrophoresis (Figure 8) and sequencing. Each of the PIV3r insertion mutants recovered contained insertions of the indicated sizes and were then evaluated for their biological properties.

EXAMPLE VII Replication of HPIV3r viruses containing GU or NCR inserts in animals and in tissue culture Multi-step growth curves The growth properties of the GU and NCR insertion mutants of PIV3r were compared to wild-type PIV3r and cp45Lr in vi tro. As shown in Figure 9, the replication rate and peak virus titer of each of the PIV3r containing any of the GU or NCR inserts was undetectable from that of wild-type PIV3r indicating that the insertion of the at least 3918 nts in length did not affect the replication of the virus in vitro.

Replication in rIVIV hamsters containing GU insertions The hamsters were inoculated intranasally with 106- TCID5o PIV3r wild type, cp45Lr or with one of the indicated mutant PIV3r carrying the GU insertions (Table 10). The nasal lungs and turbinates were harvested on day four after infection and the level of replication of each virus was determined. The insertion of GUs ranging in size from 168 nt to 1908 nt did not significantly reduce viral replication in the respiratory tract of hamsters. However, the insertion of the 3918 nt gene unit between the HN ORF and L of the wild-type PIV3 resulted in a 5 and 25-fold reduction in viral replication in the nasal and lung turbinates, respectively. This indicates that the gene unit inserts of this size are attenuating for the wild type virus whereas the shorter sizes, eg, less than about 2000 nt, have little effect on replication of the wild type virus in the respiratory tract of those of the hamsters. In this way, the length GU can be altered to determine a desired level of attenuation in the viruses for PIV vaccine.

Table 10: Replication of GU insertion mutants of PIV3r in the respiratory tract of hamsters Mean virus titer (log10 TCID5Q / g ± S. E.b) in: Virus3 Turbinates nasal Lungs PIV3r wild type 5.9 ± 0 .2 6.0 ± 0. 2 168r nt GU ins 5.9 ± 0 .1 6.4 ± 0. 1 678r nt GU ins 6.1 ± 0 .1 6.2 ± 0. 1 996r nt GU ins 5.5 ± 0 .2 5.4 ± 0. 2 1428r nt GU ins 5.9+ 0 .1 5.3 ± 0. 6 1908r nt GU ins 5.6 ± 0 .1 5.7 ± 0. 2 3918r nt GU ins 5.2 ± 0 .2 4.6 ± 0. 3 cp45Lr 3.1 ± 0 .0 1.7 ± 0. 2 1908r nt GU ins / cp45l 1.8 ± 0 .2 1.5 ± 0 to. To the hamsters, in groups of eight. they were administered 10S-0TCID5o of the virus intranasally in an inoculum of 0.1 ml. Four days later, the lungs and nasal turbinates were collected and a virus titer was determined at 32 ° C. b. HE : standard error.

As described above, the insertion of the HA gene of the measles virus in the wild-type JSr and the attenuated cp45L virus further attenuated each virus for the hamsters. Because the HA gene of the measles virus is 1936 nt in length, the effect of a gene insert of similar size (1908 nt) on the replication of cp45Lr was examined. The insertion of the 1908 gene differs from the insertion of the HA gene of the measles virus in that it can not synthesize a large polypeptide. When the insertion GU 1908 nt was combined with the amino acid substitutions of polymerase cp45L (1908r nt GU ins / cp45L in Table 10), the attenuation was increased approximately 20-fold in the upper respiratory tract. Taken together, these findings indicate that GU inserts of approximately 3918 n-lengths can attenuate a wild-type PIV3 virus for hamsters and that GU inserts of approximately half the size can further attenuate a candidate for attenuated PIV3 vaccine. In this way, GU insertions can have dual functions in the design of recombinant vaccines. This first function is to encode a protein antigen of a pathogen and the second function is to confer an attenuation phenotype.

Replication in rIVIV hamsters containing 3 'NCR inserts of the HN gene The hamsters they were inoculated intranasally with the control viruses PIV3r or the viruses carrying the insertion mutations extending the length of the 3 'NCR of the HN gene (Table 11). The lungs and nasal turbinates were harvested four days after the inoculation and the level of viral replication in each tissue was determined as described above. The NCR insertions of the HN gene range in size from 258 nt to 1404 nt without significantly reducing viral replication in the respiratory tract of hamsters (Table 3). Nevertheless, an insertion of 3126 nt affected a 16-fold reduction in viral titer in the upper and lower respiratory tracts of the infected hamsters and an NCR insert of the 3894 nt HN gene resulted in a 12-fold reduction in viral replication of the upper and lower respiratory tracts, suggesting that the increase in genome length also confers an attenuation effect on viral replication.

Table 11: Replication of NCR insertion mutants of PIV3r in the hamster respiratory tract Mean virus titre (logn) TCID50 / g ± S. E.b) in: Virus3 Turbinates nasals Lungs PIV3r wild type 6. 2 ± 0. 1 6. 4 ± 0 .1 258r nt NCR ins 5. 9 ± 0. 1 6. 5 ± 0 .1 972r nt NCR ins 5. 9 ± 0. 1 6. 6 ± 0 .1 1404r nt NCR ins 5. 9 ± 0. 2 6. 6 ± 0 .1 3126r nt NCR ins 5. 0 ± 0. 1 5. 2 ± 0 .1 3894r nt NCR ins 5. 1 ± 0. 1 5. 3 ± 0 .1 cp45Lr 3. 4 ± 0. 1 1. 9 ± 0 .2 a The hamsters, in groups of eight, were given 106- TCID50 of the virus intranasally in an inoculum of 0.1 ml. Four days later, the lungs and nasal turbinates were collected and a virus titre was determined at 32 ° C. b. S.E .: standard error.

Evaluation of the temperature sensitivity level of the GU and MCR inserts The efficiency of plating (EOP) at tolerant and non-tolerant temperatures of the PIVr in LLC-MK2 monolayers was determined as described before. (Table 12). At 32 ° C, the viruses carrying the GU insertions ranging in size from 168 nt to 3918 nt and the NCR inserts ranging in size from 258 nt to 3894 nt had a plaque morphology that was similar to that of PIV3r ts. However, at 39 ° C and at higher temperatures all the viruses carrying the insertion mutations had a small plaque phenotype. { Table 12). GU insertions ranging in size from 996 nt to 3918 nt provided viruses that were not ts at 40 ° C. However, viruses carrying the NCR inserts of HN gene of 1404 nts or higher provided viruses that were slightly ts at 40 ° C with a gradient of temperature sensitivity to provide the size of the insert. The addition of the GU 1908 nt addition to the cp45L structure provided a virus that was almost 100 times more ts at 38 ° C compared to cp45Lr, demonstrating that the ts phenotype specified by the GU 1908 nt insertion and by the ts mutations of the L gene is additive.

Table 12: Efficiency of plaque formation of GU and NCR insertion mutants of PIV3r at tolerant and non-tolerant temperatures Virus title at the indicated temperature (logi0UPF / itil) Virus 32 ° C 37 ° C 38 ° C 39 ° C 40 ° C PIV3r wild type 7. ND 7 '4 7.5 168r nt GU ins 7. ND 7. 5a 6.7a 678r nt GU ins 7. ND 7, 3a 7.0a 996r nt GU ins 7. ND 7, 0a 6.3a 1428r nt GU ins 7. ND 7, 4a 6.4a 1908r nt GU ins 7. ND 6, 5a 6.0a CM 3918r nt GU ins 6. ND 5, 7a 5.0a 258r nt NCR ins 8.1 ND 7 4a 7.5a 972r nt NCR ins 8.2 ND 7 8a 7.8a 1404r nt NCR ins 6.7 ND 5 2a < 3.7 3126r nt NCR ins 7.4 ND 6 4a 4.5 '3894r nt NCR ins 7.4 ND 5 3"5.0a Plates were enumerated by immunoperoxidase staining after incubation for 6 days at the indicated temperature. The values that are underlined and in bold type represent the lowest restriction temperature at which there was at least a 100-fold reduction in plate-forming efficiency compared to the titre at 32 ° C, which was defined as the temperature of deactivation of plate formation.

Because the insertion mutant GU 3918r nt as well as the insertion mutants NCR 3126r nt and 3894r nt replicated efficiently in vitro but replication was restricted to the respiratory tract of the hamsters, these recombinants exhibited a classification attenuation phenotype novel host. Based on the above examples, it is demonstrated that recombinant HPIV3 (HPIV3r) provides an effective vector for foreign viral protective antigens expressed as additional supernumerary genes, as exemplified by the glycoprotein hemagglutinin (HA) gene of measles virus. In another embodiment, the HPIV3r-1 antigenic chimeric virus, a recombinant HPIV3 in which the PIV3 F and HN genes were replaced by their HPIVlr counterparts provides an effective vector for the hemagglutinin-neuraminidase (HN) glycoprotein of HPIV2 in each case, the foreign coding sequence was designed and constructed to be under the control of a set of gene start and end gene transcription signals of HPIV3, inserted into the vector genome as an additional supernumous gene, and expressed as a mRNA separated by the HPIV3 polymerase. The expression of the measles virus HA glycoprotein or the HPIV2 HN from a supernumerary gene insert by the HPIV3r or HPIV3r-1 vector was determined to be stable over multiple rounds of replication. Hamsters infected with the vector HPIV3r expressing the HA vector of measles virus or HPIV3r-1 expressing the HPV2 HN glycoprotein induced an immunoprotective response for the HPIV3 and measles virus, or for HPIV1 and HPIV2, respectively. In this way, an individual HPIV3r vector expressing the protective antigen of the measles virus induced a protective immune response against two human pathogens, namely HPIV3 via an immune response to the glycoproteins present in the vector structure and the measles virus. Protective HA antigen expressed from the extra gene inserted in HPIV3r. The measles virus glycoprotein was not incorporated into the infectious HPIV3 vector virus, and therefore its expression was not expected to alter the tropism of the vector or make it susceptible to neutralization with measles virus-specific antibodies. Similarly, a simple HPIV3r-l vector expressing the protective HN antigen of HPIV2 induced a protective immune response against two human pathogens, namely HPIV1 via an immune response for the glycoproteins present in the vector structure and HPIV2 via the protective antigen. HN expressed from the extra gene inserted in HPIV3r-l.

EXAMPLE VIII A simple HPIV3r expressing up to three supernumerary foreign viral glycoproteins induces protective antibodies against the three viruses The modification of the virus for simple recombinant vaccine to induce immunity against multiple pathogens had several advantages. It is much more feasible and quick to develop a simple attenuated structure that expresses antigens against multiple pathogens than to develop a separately attenuated vaccine against each pathogen. Each pathogen offers different challenges for the manipulation, attenuation and demonstration of safety and efficacy, and it could be a frightening task to try to develop an attenuated version of each of a series of pathogens. It is also simpler and easier to prepare, manipulate and administer a virus for simple vaccine than to undertake these activities with various different attenuated viruses. The version of the vaccine virus number will also help to simplify the mandatory pediatric immunization schedule. The various attenuated viruses can be administered as a mixture, although this complicates the development of the vaccine, because each component must show to be separately safe and then be safe and effective as a mixture. A particular problem with the administration of virus mixtures is the common phenomenon of viral interference, in which one or more of the viruses in the mixture interfere with the replication of one or more of the other components. This can result in reduced replication and immunogenicity for one or more components. This common problem is made obvious by the use of a simple vector structure. Also, due to the particular safety concerns of some viruses such as for example the measles virus, it would be safer to use a comparatively benign, simple virus, such as PIV as a vector carrying multiple supernumerary antigens, as opposed to a mixture of attenuated viruses separately, each of which must be developed and validated separately. In the present example, recombinant HPIVs are constructed and shown to serve as vectors for more than one supernumerary gene with satisfactory replication and immunogenicity characteristics for the development of vaccine viruses. In particular, this example describes the design, construction, recovery and characterization of HPIV3r that express one, two or three supernumerary genes from the following list: (i) hemagglutinin-neuraminidase (HN) of HPIV1 (strain Washington / 20993 / 1964); (ii) the HN of HPIV2 (strain V9412); (iii) hemagglutinin (HA) of wild-type Edmonston strain of measles virus; and (iv) a transiently silent synthetic gene 3918-nt called gene unit (GU) (seen above). The aggregated genes were inserted into HPIV3r between the nucleoprotein (N) and phosphoprotein (P) genes, between the P genes and the membrane protein (M), or between the HN genes and the large polymerase (L). Thus, the discussion demonstrates the successful use of a modified HPIV3 vector in a recombinant for bivalent, trivalent or quadrivalent vaccine capable of inducing a multivalent immunity, for example, against the vector itself and one or two additional pathogens. Insertion of the HPIV1 and HN HN genes of HPIV2 between the N / P and P / M genes was performed as follows: the plasmid pUCI19 (A // II NP), a subclone of the HPIV3 antigenomic cDNA (Durbin, J. Virol. 74_: 6821-31, 2000, incorporated herein by reference), was modified by site-directed mutagenesis to insert a unique A // II site into (i) the coding region in the 3 'direction of the N gene of HPIV3 (CTAAAT to CTTAAG, HPIV3 nts 1677-1682), or (ii) the non-coding region in the 3 'direction of the P gene of HPIV3 (TCAATC to CTTAAG, HPIV3 nts 3693-3698). Each A // II site was then modified by inserting a duplex oligonucleotide, creating the intermediate plasmids pUC (GE / GS-N-H) N-P and pUC (GE / GS-N-H) p_M, respectively. The inserted duplex contained a gene end sequence (GE) -of HPIV3, the conserved intergenic trinucleotide (IG) sequence and a gene start sequence (GS) of HPIV3, which are cis-acting signals that direct the termination and direct transcriptional initiation, respectively (Figure 10). The additional, unique restriction endonuclease sites were included in the multiple cloning region to facilitate subcolonation and subsequent selection, including the Ncol and HindIII sites for the addition of the HN ORFs of HPIV1 and HPIV2. In this way, a foreign ORF inserted in the multiple cloning site could be under the control of a set of HPIV3 transcription signals and could be expressed as an mRNA separated by HPIV3 polymerase. The multiple cloning site also contained an MluI site to insert oligonucleotides of varying lengths as necessary to make the entire inserted sequence conform to the six rule (Calain et al., J. Virol. 67: 822-30, 1993; Durbin et al., Virology 234: 74-83, 1997b; 1999a Skiadopoulos et al., Virology 272: 225-34, 2000). The HN ORF of HPIV1 available as a Ncol to HindIII restriction fragment from p38'A31hc # 6 (Tao et al., J. Virol. 72: 2955-2961, 1998), was inserted into the Ncol sites to ffindIII of pUC ( GE / GS-NH) Np and pUC (GE / GS-NH) P_M to generate pUC 1HNN-P and pUC IH pM, respectively. The short duplex oligonucleotides were inserted into the unique MluI restriction site to adjust the sequence to conform to the six rule. These chimeric subgenomic cDNAs were then cloned into the full length HPIV3 antigenic cDNA p3 / 7 (131) 2G +, referred to herein as pFLC HPIV3 wild type, to provide pFLC HPIV3 lHNN_p and pFLC HPIV3 1HNP-M, respectively (Figure 11, plasmids from which the second and third recombinant viruses were isolated from the top). The HPIV2 HN ORF, available within an N-to-III restriction fragment of p32Hnhc # 3 31hc (Tao et al., J. Virol., 72; 2955-2961, 1998, incorporated herein by reference), was inserted into the Ncol to JTindIII sites of pUC (GE / GS-HN) Np and pUC (GE / GS-HN) pM to generate pUC 2HNN-p and pUC 2HNp_M, respectively. The short oligonucleotide duplexes were inserted into the unique MluI restriction site to adjust the sequence to conform to the six rule. Inadvertently, the inserted oligonucleotide was a shorter nucleotide than required to specify that the genome of the recovered virus could conform to the six rule. Therefore, all the cDNAs carrying the insertion of the HIV2 HN gene did not conform to the six rule. However, the virus recovered from each of these cDNAs. These chimeric subgenomic cDNAs were cloned into the full-length PI3 antigenomic wild-type HPIV3 pFLC cDNA to provide pFLC PIV3 2HN [N_p) and pFLC PIV3 2HN < P-M), respectively (Figure 11, the plasmids from which the fourth and fifth recombinant viruses were isolated from the top). Additional recombinant HPIV3 antigenomic cDNAs were assembled to contain up to three supernumerary foreign genes in various combinations and locations in the HPIV3 structure (Figure 11). These antigenomic cDNAs were assembled from the subgenomic cDNAs described above in which the HPIV1 or HPIV2 HN was inserted between the N and P genes or the P and M genes. Other subclones used for the assembly contained the HA gene of the measles virus between P / M genes or NH / L genes as described above. Another subclone used in the assembly contained the GU 3918-nt between the HN and L genes, as described above. The recombinants containing two to three supernumerary inserts were as follows: HPIV3r 1HNN-P 2HNp_M (Figure 11, sixth recombinant from the top) contained the HN HPIV1 or HN HPIV2 genes inserted between the N / P and P / M genes , respectively; HPIV3r 1HNN_P 2HNP-M HAHN-L (Figure 11, seventh recombinant) contained the HN of HPIV1, HN of HPIV2 and the HA of the measles virus inserted between the genes N / P, P / M, and HN / L, respectively; and PIV3r 1 ??? -? 2HNp_M 3918GUHN-L (Figure 11, bottom), contained, the HN genes of HPIV1 and HN of HPIV2 inserted between the N / P and P / M genes, respectively, and furthermore contained the GU insert 3918-nt between the HN genes and L. It is remarkable that the penultimate of these constructions, HPIV3r 1HNN-P 2HNP-M (Figure 11, seventh construction of the upper part), contained protective antigens for four pathogens: HPIV3 (HN and F), HPIV1 (HN), HPIV2 (HN) and the measles virus (HA). The total length of the foreign sequence inserted in this recombinant was approximately 5.5 kb, which is 36% of the total HPIV3 genome length of 15.462 nt. The last recombinant, HPIV3r-lHNN-p2HNp-MGUHN-L (Figure 11, bottom), was approximately 23 kb in length. This is 50% greater than wild-type HPIV3, and greater than any biologically or recombinantly derived paramyxovirus described above.

In vitro recovery and replication of recombinant HPIV3 carrying one, two or three inserts of supernumerary gene The full-length HPIV3 antigenic cDNAs carrying single or multiple supernumerary genes of the heterologous paramyxovirus protective antigens were separately transfected into HEPA monolayer cultures. 2 in six-well plates (Costar, Cambridge, MA) together with the support plasmids pTM (N), pTM (P no C), and pTM (L) and LipofectACE (Life Technologies, Gaithersburg, MD) and the cells were infected simultaneously with MVA-T7, a virus recombinant for replication-defective vaccine that encodes the bacteriophage T7 polymerase protein using the techniques described above (Durbin et al., Virology 235: 323-332, 1997a; Skiadopoulos et al., Virology 272: 225-34, 2000, each incorporated herein by reference). After incubation at 32 ° C for up to four days, the transfection collection was run in LLC-MK2 monolayer cultures in a 25 cm2 flask and the cells were incubated for 5 days at 32 ° C. Virus recovered from the cell supernatant was further passaged on LLC-MK2 cells at 32 ° C to amplify the virus. HPIV3r carrying individual or multiple foreign gene inserts were biologically cloned by plaque purification on LLC-MK2 cells as described above (Skiadopoulos et al., J. Virol. _3: 1374-81, 1999a, incorporated herein by reference). reference). Viral suspensions derived from biologically cloned viruses were amplified in LLC-MK2 cells and yielded final titers of 107 and 109 TCID50 / ml, similar to the range of titers typically obtained by wild-type HPIV3r. The recombinant viruses were analyzed for their growth capacity at 39 ° C. Surprisingly, several HPIV3r carrying individual or multiple foreign gene insertions (HPIV3r 1HNN-P, HPIV3r lHNN_p2HNp-MHAHN-L and HPIV3r 1HNN-P 2HNp_m 3918 GUHN-L) were restricted from 100 to 1000 fold for replication at 39 ° C compared to the replication at the tolerant temperature. Viral RNA (vRNA) was isolated from biologically cloned recombinant chimeric viruses as described above (see also, Skiadopoulos et al., J. Virol. 73: 1374-81, 1999a, incorporated herein by reference). This was used as the template for reverse transcription and polymerase chain reaction (RT-PCR) that uses specific primers bordering the insertion sites. The amplified products were analyzed by restriction endonuclease digestion and partial DNA sequencing of the binding regions to confirm the presence and identity of each foreign insert. In all cases, the expected correct sequence was confirmed.

Replication in the respiratory tract of HPIV3r hamsters expressing one, two or three supernumerary foreign protective antigens It was previously shown that HPIV3r or HPIV3r-l viruses expressing a supernumerary viral protective antigen gene were replicated efficiently in vitro and in vitro. alive and induced protective immune responses against the vector virus and the virus represented by the supernumerary antigen gene. However, it is unknown whether an HPIVr could adapt to one or more supernumerary genes and retain the ability to efficiently replicate in vitro and in vivo and induce protective immune responses for both the vector and expressed supernumerary antigens. The present example indicates that this is indeed possible. The hamsters in groups of eight were inoculated intranasally with 106 TCID50 of each HPlV3r carrying individual or multiple supernumerary foreign gene inserts or with the control viruses (Table 13). Nasal turbinates and lungs were collected four days after infection and the virus present in tissue homogenates was quantified by serial dilution on LLC-MK2 monolayer cultures at 32 ° C as described above (see also, Skiadopoulos et al., J. Virol. 73: 1374-81, 1999a). The virus was detected by hemadsorption with guinea pig erythrocytes and the virus titre average for each group was expressed as logio TCID50 (50% infectious dose of tissue culture / tissue gram ± SE).

Table 13 Replication of recombinant HPIV3 containing individual or multiple supernumerary gene inserts expressing the HPIV1, HPIV2 virus glycoprotein genes or measles virus in the upper and lower respiratory tract of hamsters Group3 Virus0 Virus titer of virusc (logio TCID50 / g ± SE) in: No. Turbinates Lung Reduction Nose reduction title (logio) d title (log10) d 1 HPIV3r ??? ,, - ?, 4.5 ± 0.2 1.8 3.9 ± 0.2 3.0 2 HPIV3r ???,? - ,,) 3.5 ± 0.2 2.8 4.3 ± 0.2 2.3 3 HPIV3r 2HN (Np, 5. ± 0.2 0.9 5.3 ± 0.3 1.6 4 HPIV3r 2HN (pM) 6.3 ± 0.1 0.0 6.3 ± 0.5 0.6 5 HPIV3r HA (Kp) 5.3 ± 0.2 1.0 5.8 ± 0.4 1.1 6 HPIV3r HA (pM) 6.0 ± 0.2 0.3 7.3 ± 0.2 -0.4 7 HPIV3r (HN- L) 6.0 ± 0.1 0.3 6.6 ± 0.2 0.3 S HPIV3r ???,? - ?, 2HNtP.H) 5.2 ± 0.1 1.1 5.0 ± 0.3 1.9 9 HPIV3r 1HN (Np) 2HN (MP) HA (HN-LJ 1.6 ± 0.1 4.7 2.5 ± 0.1 4.4 10 HPIV3r lHN (IJ.p) 2HN (NP) 3918 GU, "» -_,) 2.0 ± 0.3 4.3 1.8 ± 0.2 5.1 11 HPIV3r cp45 4.6 ± 0.1 1.7 2.1 ± 0.2 4.8 12 HPIV3r wild type 6.3 ± 0.1 - 6.9 ± 0.1 -a. 8 hamsters per group. b. Each hamster was inoculated with 106 TCIDso virus in an inoculum of 0.1 ml c. The virus was titrated by serial dilution on LLC-MK2 monolayer cultures at 32eC. d. The reduction in virus replication was compared with wild-type HPIV3r (group 12) It was previously shown that a HPIV3r expressing Ha from the measles virus from a supernumerary gene insert between the HN and L genes, between the N and P genes, or between the P and M genes of HPIV3 was modestly restricted (approximately 10 to 20 times) in the replication in the upper and lower respiratory tract of the hamsters. This was confirmed in the present experiment, in which HPIV3r containing measles virus HA as an individual supernumerary gene between the N / P, P / M or HN / L genes was attenuated up to 10 times (Table 13, Groups 5, 6 and 7). Similarly, insertion of the HPIV2 HN gene between the N and P genes or between the P and M genes of HPIV3 also exhibited a modest (approximately 10 to 20 fold) reduction in replication in the respiratory tract of hamsters (groups 3 and 4 of Table 13). In contrast, insertion of the HN gene of HPIV1 between the P and M genes or between the N and P genes resulted in more than approximately 100 fold reduction of replication in the upper and lower hamster respiratory tract (groups 1 and 2 of table 13). Because the HA gene insertions of HPIV1 HN virus, HPIV2 HN, and measles, are all of approximately the same size (1794 nt, 1781 nt and 1926 nt, respectively), this was not likely to be due to the length of the insert. Therefore, the higher level of attenuation conferred by the introduction of the HN gene of HPIV1 is probably due to an additional attenuation effect that is specific for the expression of the HN protein of HPIV1 in the HPIV3 vector replication. Thus, in some cases, such as with the HPIV1 HN, a supernumerary antigen can attenuate HPIV3r for hamsters prior to and beyond the modest attenuation due to the insertion of an additional gene. Inspection of the data in Table 13 indicates that the insertion site also plays a role in the level of replication restriction of the chimeric HPIV3r in the hamster respiratory tract. The insertion of the Ha gene of the measles virus or the HN gene of HPIV2 between the N and P genes of HPIV3r resulted in a greater reduction of replication in the upper and lower respiratory tract of hamsters than that of the insertion between the P genes. and M. { Table 13, compare groups 3 against 4 and 5 against 6). This site-specific attenuation effect on HPIV3 vector replication was not evident for HN gene insertions of HPIV1, presumably because it was masked by the more substantial attenuation effect specific to HN of HPIV1. Chimeric HPIV3r recombinant viruses exhibited an attenuation gradient that was a function of the number of inserts of the supernumerary gene. These viruses that carry three aggregated genes exhibited the greatest effect, and were reduced approximately 10,000 to 108,000 times in replication in the upper and lower respiratory tract of infected hamsters (Table 13, groups 9 and 10). The chimeric HPIV3r recombinant virus carrying two gene inserts exhibited an intermediate attenuation level and was reduced approximately 12 to 80 fold (Table 13, group 8). Chimeric HPIV3r recombinant viruses carrying a supernumerary gene (except those carrying the HN gene of HPIV1) were reduced only approximately 10 to 25 times (groups 3-7 in Table 13). Importantly, recombinant HPIV3r chimeric viruses carrying one, two or three inserts of supernumerary gene were replicated in all animals. The most attenuated of these viruses, namely those carrying three supernumerary genes, were practically more attenuated than cp45r (group 11) with respect to replication in the upper and lower respiratory tract. Immunogenicity in hamsters of HPIV3r that express one, two or three supernumerary foreign protective antigens The hamsters were infected with wild type HPIV1, wild type HPIV2, wild type HPIV3r or HPIV3r carrying individual, double or triple supernumerary gene inserts as described above. Serum samples were collected at 3 days prior to immunization and at 28 days after immunization and analyzed for HPIV1, HPIV2, HPIV3 or measles virus-specific antibodies by virus neutralization analysis specific for either HPIV1 virus or of measles, or by haemagglutination-inhibition analysis (HAI, for its acronym in English) for specific antibodies HN of HPIV3 or HPIV2 (Table 14). All HPIV3r viruses produced an important immune response for the HPIV3 structure with the exception of viruses carrying all three supernumerary gene insertions. Reduced or absent immune response in hamsters infected with any of the HPIV3r 1HNN_P 2HNN-p HAHN-L, or HPIV3r 1HNN.P 2HNN-P 3918GUHN-L, was probably a result of these viruses that are too attenuated for replication in hamsters. Likewise, the immune response for the vectorized antigens in the viruses carrying three foreign genes was also little or undetectable. In contrast, viruses carrying an individual or double stranded gene insertion induced an immune response against each of the additional antigens, demonstrating that the vectorized foreign genes are immunogenic in hamster and in the example of HPIV3r lHNN_ p 2HNN _p (Table 14; group 11) can be used to induce an important immune response for the three different viruses: HPIV1, HPIV2 and HPIV3.

Table 14 Immune response in hamsters to immunization with HPIV3r vectors expressing individual or multiple supernumerary protective antigens of HPIV1, HPIV2, or measles virus3 Virus Group Serum antibody titre "(mean log2 ± S.E.) for the indicated virus No. HPIV3C HPIVla HPIV2e Measles Virus £ 1 HPIV3r wild type 10.0 ± 0 - - - 2 HPIV2 wild type < 2.0 ± 0 - 8.0 ± 0.0 - 3 HPIV1 wild type < 2.0 ± 0 5.4 ± 0.3 - - 4 HPIV3r ?? (? -) 9.5 ± 0.2 - - 12.4 ± 0.4 5 HPIV3r 8.7 ± 1.4 - - 11.8 ± 0.2 6 HPIV3r 9.0 ± 0 - 8.1 ± 0.6 7 HPIV3r lH (N.pj 9.5 ± 0.2 3.4 ± 0.6 - - 8 HPIV3r ???,? -?) 7.2 ± 0.8 2.7 ± 0.3 - - 9 HPIV3r 2HN (li-p) 9.8 ± 0.5 - 9.3 ± 0.8 - 10 HPIV3r 2HN (p. ", 10.0 ± 0.5 - 8.3 ± 1.1 - 11 HPIV3r 1HN (NP) 2HN (Np, 9.6 ± 0.7 5.5 ± 0.4 8.3 ± 0.8 - 12 HPIV3r lHN (Np) 2HN (Np) HA (HI) <2.0 ± 1.0 1.0 ± 0.3 <2.0 ± 0.0 <3 13 HPIV3r lHN, Np) 2HN (NP) 3918GU (HH-lí 4.3 ± 0.7 2.3 ± 0.6 < 2.0 ± 0.0 - 14 HPIV3r cp45 7.7 + 0.2 - - -a Average antibody response in hamster groups (n = 6 ) inoculated intranasally with lO ^ CIDso HPIV3r expressing the hemagglutinin-neuraminidase protein of HPIV1 (1HN), HPIV2 (2HN) or hemagglutination of measles virus (HA) inserted between the N and P (NP) genes, the P and M genes (PM) or the HN and L genes (HN-L) of HPIV3r b The sera were collected 3 days before and 28 days after the immunization c) Average antibody titer for haemagglutination inhibition (HAI) for HPIV3 d) Neutralization antibody titre average for HPIV1, HAI antibody titre average for HPIV2, F. Neutralizing antibody titre for measles virus (60% reduction in plaque, PRN).

EXAMPLE IX Use of HPIV3r-NB as an attenuated vector for the measles virus HA protein The use of an animal virus that is attenuated in humans due to a host classification restriction as a vaccine against an antigenically related human counterpart is the basis of the Jennerian proposal for the development of the vaccine. The Kansas (Ka) strain of bovine parainfluenza virus type 3 (BPIV3) was found to be restricted from 100 to 1000 fold in replication in rhesus monkeys compared to the type 3 human parainfluenza virus (HPIV3), and was also shown be attenuated in humans (Coelingh et al., J. Infect. Dis. 157: 655-62 / 1988; Karron et al., J Infect. Dis. 171: 1107-14, 1995b, each incorporated herein by reference). A type 3 viable chimeric recombinant human parainfluenza virus (HPIV3) was previously produced which contained the nucleoprotein (N) open reading frame (ORF) of Ka of BPIV3 instead of the ORF N of HPIV3. This chimeric recombinant was previously designated Kac-N (Bailly et al., J. Virol. 74: 3188-3195, 2000a, incorporated herein by reference) and referred to herein as HPIV3r-NB. This previous study was initiated with an exchange of ORF N because, among the PIV3 proteins, the N protein of BPIV3 and HPIV3 possesses an intermediate level of amino acid sequence identity (85%) (Bailly et al., Virus Gene 20). : 173-82, 2000b, incorporated herein by reference), and it was shown that this recombinant N of BPIV3 / HPIV3 is viable (Bailly et al., J. Virol. 74_: 3188-3195, 2000a, incorporated herein) as reference) . This represents a proposal "Modified Jennerian" in which only a subset of genes in the vaccine virus was derived from the animal counterpart. HPIV3r-NB grew to a titer comparable to HPIV3r and BPIV3 precursor viruses in LLC-MK2 monkey kidney and Madin Darby bovine kidney cells (Bailly et al., J. Virol. 74 ^ 3188-3195, 2000a). In this way, the heterologous nature of the N protein did not prevent the replication of HPIV3r-NB in vitro. However, HPIV3r-NB was restricted in replication in rhesus monkeys to a degree similar to that of its BPIV3 precursor virus (Bailly et al., J. Virol. 74: 3188-3195, 2000a). This identified protein N of BPIV3 as a determinant of the host classification restriction of BPIV3 replication in primates. The HPIV3r-NB chimeric virus thus combines the antigenic determinants of HPIV3 with the host classification restriction and the attenuation phenotype of BPIV3. There are 79 differences of a total of 515 amino acids between the N proteins of HPIV3 and BPIV3 (Bailly et al., Virus Genes 20: 173-82, 2000b). Many of these 79 amino acid differences probably contribute to the host classification attenuation phenotype of HPIV3r-NB. Because the host classification restriction is anticipated to be based on many amino acid differences, it is anticipated that the attenuation phenotype of HPIV3r-NB will be genetically stable even after prolonged replication in vivo. Despite its restricted replication in rhesus monkeys, HPIV3r-NB induced a high level of resistance to inoculate the monkeys with wild-type HPIV3 (wt) and this level of resistance was imperceptible from that conferred by immunization with wild-type HPIV3r. The infectivity, attenuation and immunogenicity of HPIV3r-NB suggests that this novel chimeric virus is an excellent candidate as an HPIV3 vaccine (Bailly et al., J. Virol., 14: 3188-3195, 2000a). In addition, as described below, it is shown herein that these chimeric viruses are excellent candidates to serve as an attenuated vector for the expression of supernumerary protective antigens, such as the HA of measles virus. The vector component of the resulting chimeric virus induces an immune response against HPIV3, and the aggregated supernumerary genes induce immune responses against their respective heterologous pathogens. In this specific example, a virus is produced for divalent attenuated vaccine that simultaneously induces immune response to HPIV3 and measles virus. It was previously shown that HPIV3r can be used as a vector for the expression of the hemagglutinin protein (HA) of the measles virus. In two examples, the attenuated vectors of cp45Lr HA (N-P) and cp45r HA (HN-L) expressing the Ha gene of measles virus proceeded three point mutations of attenuating amino acid in the structure of the vector. The HPIV3r-NB vector of the present invention will probably be even more stable than vectors having an attenuation phenotype based on three point amino acid mutations. Also previously, it was shown that the insertion of HA as a supernumerary gene in HPIV3r conferred some attenuation in the replication of both wild type HPIV3 and attenuated HPIV3cp45L for hamsters. In addition, the insertion of a 1908-nt gene insert in HPIV3 did not attenuate the wild-type structure, although it increased the attenuation level of a structure carrying the cp45L mutations for replication in hamsters. Therefore, insertion of the HA gene of measles virus into the host-restricted restricted HPIV3r-NB virus was protected to further attenuate its growth in vitro and / or in vivo. Inserts that affect replication in vitro or in vivo can be problematic for the development of specific vaccines such as HPIV3r-NB.

Specifically / a candidate virus that is quite restricted in in vitro replication could be difficult to manufacture, and one that is quite restricted in in vivo replication could be over-attended and not be useful as a vaccine. It is also not known if the HPIV3r-NB chimeric virus expressing the measles virus HA glycoprotein, designated HPIV3r-NBHA (pM), could be satisfactorily immunogenic in primates against both HPIV3 and measles viruses because all previous studies with HPIV3 expressing HA were conducted in a rodent model. The present example, which details the generation of HPIV3r-NB HA (p_M) using reverse genetic techniques, indicates, surprisingly, that the insertion of the Ha gene in does not further restrict its replication in rhesus monkeys. Presumably, the effect of attenuation of the insertion is masked by the genetic elements present in the NB gene that specify the host classification restriction of the replication in primates. Instead, the HPIV3r-NB HA (p-M) is successfully attenuated in rhesus monkeys. Immunization of rhesus monkeys with HPIV3rNB HA (P-.M) induced a replication resistance of the wild-type HPIV3 inoculated virus and stimulated high levels of neutralizing antibodies to the measles virus, levels that are known to be protective in humans (Chen et al., J. Infect. Dis. 162: 1036-42, 1990, incorporated herein by reference).

The construction of a pFLC HPIV3-NB HA (PM), a chimeric bovine / human PIV3 antigenomic cDNA, coding for the ORF of the N gene of BPIV3 instead of the ORF of the N gene of HPIV3r and the HA gene of the virus of Measles as a supernumerary gene inserted between the P and M genes of HPIV3r The full length antigenomic cDNA plasmid pFLC HPIV3-NBHA (PM) (Figure 12) was constructed in two steps. First, the pLeft-NB plasmid constructed above contains the 3 'half of the HPIV3 antigenic cDNA (HpIV3 nts 1-7437, the last position being an Xhol site within the HN gene) with the ORF N of HPIV3 replaced by that of BPIV3 ( Bailly et al., J. Virol, 74: 3188-3193, 2000a, incorporated herein by reference). The PshAI-NgoMIV fragment was excised from this plasmid. Note that the PshAI site at position 2147 in the HPIV3 antigenome sequence (see Figure 12) and the NgoMIV site is presented in the vector sequence and thus eliminates all HPIV3 sequences in the 3 'direction of the PshAI site. This fragment was replaced by the PshAI-NgoMIV fragment from the plasmid pLeftHA | P_M) constructed above, which contains the ORF HA of the measles virus under the control of the HPIV3 transcription signals and was inserted between the N and P genes of (Durbin , J. Virol. 74: 6821-31, 2000, incorporated herein by reference). This provided pLeft-NBHAP-M. Next, the WgoMIV fragment for Xho I 11899 nt of pLeft NB HAp-M, containing the 3 'half of the HPIV3 antigenic cDNA including the N gene ORF of BPIV3 and the insert of the HA gene of measles virus, was cloned into the NgoMIV window for Xho I of pRight, a plasmid encoding the 5 'half of the HPIV3 antigenomic cDNA (PIV3 nts 7462-15462) (Durbin et al, Virology 235: 323-332, 1997a). ] this provided pFLC HPIV3-NB HAP.M, a plasmid carrying the full-length antigenomic cDNA of HPIV3 containing the ORF N of BPIV3 in place of the ORF N of HPIV3 and containing the Ha gene of the measles virus as a gene supernumerary inserted between the P and M genes of HPIV3.

Recovery of the chimeric HPIV3r expressing the bovine N gene and the HA gene of the measles virus The HPIV3r-NB HAP_M was recovered from HEp-2 cells transfected with pFLC HPIV3-NB HAP_M. To accomplish this, the pFLC HPIV3-NB HAP-M was transfected into HEp-2 cells in six-well plates (Costar, Cambridge, MA) together with the support plasmids pTM (N), pTM (P no C) and pTM (L) and LipofectACE (Life Technologies, Gaithersburg, MD), and the cells were simultaneously infected with MVA-T7, a virus recombinant for replication-defective vaccine that codes for the bacteriophage T7 polymerase protein, as described above. After incubation at 32 ° C for four days, the transfection collection was passed over LLC-MK2 cells in a delay of 25 cm 2 and the cells were incubated for 5 days at 32 ° C. The recombinant virus from the cell supernatant was amplified by an additional step on LLC-K2 cells at 32"C. HPIV3r-NB HAP-M was biologically cloned by plaque purification on LLC-MK2 monolayer cultures as described above. derived from the biologically cloned virus was amplified in LLC-MK2 monolayer cultures at 32 ° C. Viral RNA (vRNA) was isolated from the biologically cloned recombinant chimeric viruses as described above.RTM PCR was performed using pairs of specific oligonucleotide primers that Extend the ORF N of BPIV3 or the Ha gene of the measles virus and the amplified cDNAs were analyzed by restriction endonuclease digestion and partial DNA sequencing as described above.This confirmed the presence of the replacement of ORF N BPIV3 and the insert of the measles virus supernumerary gene Ha The expression of the measles virus HA protein confirmed initially by immunostaining plates formed on LLC-K2 monolayer cultures infected with HPIV3rNB HAP_M using mouse monoclonal antibodies specific for the Ha protein of measles virus and antibodies conjugated with goat anti-mouse peroxidase, as previously described (Durbin, J Virol. 7_4: 6821-31, 2000, incorporated herein by reference).

HPIV3r-NB HAp-M replicates at the same level as HPIV3r-NB in the respiratory tract of rhesus monkeys It was subsequently determined whether acquisition of the HA insert of measles virus significantly decreased the replication of HPIV3r-NB in the upper and lower respiratory tract , as observed when a supernumerary gene was inserted into an attenuated HPIV3 structure lacking a bovine chimeric component. It was also determined whether HPIV3r-NB HAp-M sufficiently immunized induces an immune response against both HPIV3 and measles viruses in vivo. Replication of HPIV3r-NB HAp-M in the upper and lower respiratory tract of rhesus monkeys was compared to that of their HPIV3r-NB precursor as well as wild type HPIV3 and wild type BPIV3 (Table 15). Rhesus monkeys that were seronegative for both HPIV3 and measles viruses were simultaneously inoculated intransally (IN) and intratracheally (IT) with one milliliter per site of L15 medium containing 105 TCID50 virus suspension, as described above (Bailly et al., J. Virol. 74: 3188-3195, 2000a). Samples were collected by a nasopharyngeal (NP) swab on days 1 to 10 • after infection, and samples were collected by tracheal wash (TL) on days 2, 4, 6, 8 and 10 after infection. The virus present in the NP and TL specimens was quantified by serial dilution in cell monolayers. LLC-MK2 at 32 ° C and the title obtained was expressed as log 10 TCID 50 / ml (Table 15). This comparison showed that the chimeric virus HPIV3r-NBHA (p-M) 3-1 same level in the upper and lower respiratory tract of the rhesus monkey as its HPIV3r-NB precursor virus. This level of replication was also comparable with that of the BPIV3 precursor virus, demonstrating that HPIV3r-NB HA (PM) retains the attenuation phenotype of HPIV3r-NB and BPIV3 and, unexpectedly, that the insertion of the HA gene of the measles virus in the genome HPIV3r-NB does not additionally attenuate this virus for replication in the respiratory tract of rhesus monkeys.

Table 15 A human / bovine chimeric PIV3 containing the hemagglutinin gene of measles virus is successfully attenuated for replication in the upper and lower respiratory tract of rhesus monkeys, induces antibodies to the vius of both HPIV3 and measles and protects against inoculation of wild-type HPIV3 virus Response to immunization Response to inoculation Response to wild-type HPI3 in the 28 or 31 administration of the measles virus vaccine (Mor a ten) on day 59 Replication of the Response of antibody Replication of the Response Serum virus response serum antibody antibody serum serum Media of the title Title of Media Mean of the Mediated Title of the virus picoc antibody title of the virus antibody title of (log2 ± TCID50 / ml ± serum HAZ HPl3g antibody HAI (media antibody (media for the reciprocal (log10TCIDS0 / ml ± SE) for the EscobiLavirus rec proca virus Escobi- log2 Wash ± SE) Llon measles measles NP 116 ± tracheal log2? NP tracheal for HPIV3 (60% PRN, S.E.) for (60% PRN, on average day HPIV3 on average 56/59"reciprocal day 28 / 31d'e reciprocal log2 ± SE) (day 87 log2 ± SE ) after the Group Virus No. (day 31 first No immunizes after immunization) f * s "immunization) f HPIV3r ts 4.9 ± 0.4 3.2 ± 0.6 9.3 ± 0.6 <5.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.0 12.0 ± 0.0 8.2 ± 0.8 2 HPrv3r- B 2.6 ± 0.6 2.0 ± 0.4 7.3 ± 0.3 < 5.5 ± 0.0 1.4 ± 0.9 0.5 ± 0.0 9.0 ± 1.0 10.1 ± 0.4 3 HPIV3r-t½ 2.2 ± 0.6 2.8 ± 0.6 6.8 ± 0.3 9.6 ± 0.5 1.2 ± 0.7 2.3 ± 0.2 11.5 ± 0.3 10.2 ± 0.4 4 BPTV3 Ka 2.3 ± 0.2 1.9 ± 0.2 5.0 ± 0.4 ND 2.9 ± 0.3 2.0 ± 0.5 11.5 ± 0.3 ND 5 nob ND ND < 2 ND 4.5 ± 0.3 4.5 ± 0.2 12.0 ± 0.6 ND The present study included 4 monkeys that received HPIV3r-NB HAIt > -M) and two monkeys in each of the groups that received HPIV3r wild type, HPIV3r-NB or Ka of BPIV3. With the exception of the group that received HPIV3r-NB HA (p-M), the presented data include historical data from studies reported in Bailey et al., J. Virol. 74: 3188-3195, 2000, and Schmidt et al., J. Virol. 74: 8922-8929, 2000. Historical data of Schmidt et al., J. Virol. 74: 8922-8929, 2000. The monkeys were inoculated intranasally and intratracheally with 10 TCID50 of virus in an inoculum of 1 ml at each site. Samples were collected by nasopharyngeal swab on days 1 to 10 after infection. Samples were collected by tracheal lavage (TL) on days 2, 4, 6, 8 and 10 after infection. The average peak virus titers for each animal in your group without taking into account the sampling day. HE = standard error. Virus titrations were performed on LLC-MK2 cells at 32 ° C. The limit of detection of the virus titer was 10 TCID5o / ml. In the present study monkeys sera were collected on day 31 after immunization and the animals were then inoculated with HPIV3. In the two previous studies, the monkeys were sampled and inoculated on day 2T after immunization, the sera collected for the present study and the two previous studies They analyzed at the same time. The serum HAI titer was expressed as the reciprocal mean log2 ± standard error, SE. The animals were immunized on day 59 with 106 pfu of the Moraten vaccine strain of measles virus administered parenterally (IM). The serum was collected 28 days later (ie, 87 days after the first immunization). The data shown were obtained from samples collected only from animals in the present study. The mean neutralizing antibody titer for wild-type measles virus is expressed as the mean reciprocal log2 standard error. PNR, plate reduction neutralization. From 28 to 31 days after the immunization the monkeys were inoculated intranasally and intratracheally with wild type HPIV3 or TCID3 in an inoculum of 1 ml at each site. The NP and TL samples were collected on days 0, 2, 4, 6 and 8 after inoculation. The titles obtained for the NP and TL samples on day 0 were < 2.0 logio TCID5o / ml. With the exception of group 5, the data shown is from the present study.

Immunization of rhesus monkeys with HPIV3r-NB HA < p-M) induces high antibody titers against both HPIV3 and measles viruses and protects monkeys from inoculation with HPIV3 rhesus monkeys immunized with HPIV3r-NB HAp-M developed high levels of serum antibodies against both HPIV3 and measles viruses (Table 15). Serum HPIV3 antibodies were quantified by haemagglutination inhibition analysis (HAI) using guinea pig erythrocytes as described previously (Durbin, J. Virol. 74: 6821-31, 2000, incorporated herein by reference), and the titers they were expressed as the reciprocal mean log2 + SE. High levels of serum HAI antibodies for HPIV3 were induced by both HPIV3r-NB HAP-M and HPIV3r-NB, demonstrating that these attenuated recombinants can induce an important immune response against the structure antigens of the HPIV3 vector. It was also found that rhesus monkeys immunized with HPIV3r-NB HAP-M developed high levels of measles virus neutralizing antibodies, serum, 31 days after immunization, levels that are in excess of those needed to protect humans against infection with measles virus (Chen et al., J. Infect. Dis. 162: 1036-42, 1990, incorporated herein by reference). Neutralizing antibody titers were placed against the wild-type measles virus were quantified as described previously (Durbin, J. Virol 74: 6821-31, 2000), and titers were expressed as the reciprocal mean log2 ± SE (Table 15). To compare infection capacity with candidates for HPIV3r-NB HAP-M and HPIV3r-NB virus vaccine attenuated in vivo to protect against wild type HPIV3, monkeys were inoculated IN and IT with 106 TCID5o of wild type HPIV3 31 days later of the initial infection (Table 15). Samples were collected by nasopharyngeal brush and tracheal wash on days 2, 4, 6 and 8 after inoculation. The virus present in the specimens was quantified by serial dilution on LLC-MK2 monolayer cultures as described above. HPIV3r-NB HAP.M and HPIV3r-NB conferred a comparable high level of protection against inoculation with wild-type HPIV3 as indicated by a 100 to 1000-fold reduction in replication of wild-type HPIV3 in the respiratory tract of monkeys immunized. This demonstrates that the insertion of the HA gene of the measles virus in the chimeric human / bovine PIV3 does not decrease the level of protection induced by the HPIV3 glycoproteins present in the structure of the attenuated virus vector. The immunogenicity of HPIV3r-NB HAP_M was then compared to that of the authorized Moraten strain of the measles virus vaccine attenuated in vivo in rhesus monkey, a species in which both the PIV3 virus and the of measles are reapplied efficiently. Rhesus monkeys previously infected with an HPIV3r virus or with HPIV3r-NB HAP-M were immunized parenterally (I) with 106 pfu of the Moraten strain of the attenuated measles virus vaccine in vivo on day 59 and serum samples were taken in on day 87 and analyzed for neutralizing antibodies against the measles virus (Table 15). In animals that were not affected by the measles virus before receiving the Moraten vaccine (Table 15, groups 1 and 2), the titre of specific antibodies for measles induced by the Moraten vaccine was approximately the same as that observed in animals immunized for HPIV3r-NB HAP-M (Table 15, group 2). In this way, the HPIV3r-NB HAP_M vector expressing the HA glycoprotein measles virus was equivalent to the Moraten vaccine in the ability to induce neutralizing antibodies to the virus in this primate model. An important advantage of HPIV3r-NB HAP_M as a vaccine for measles virus with respect to the Moraten vaccine is that the PIV vector can be administered via the intransal route, whereas vaccines for the attenuated measles virus in vivo are not infectious consistently along this route, probably as a consequence of its attenuation and adaptation to cell culture. This makes it possible to immunize with HPIV3r-NB HAP_M in early childhood, an age group that can not be immunized with an attenuated live measles virus vaccine in vivo such as the Moraten strain due to the neutralizing and immunosuppressive effects of the antibodies maternal (Durbin, J. Virol. 74: 6821-31, 2000, incorporated herein by reference). Other advantages are also described above, which include the superior development of the PIV vector in the cell culture and the lack of incorporation of the measles virus HA into the virions, which must prevent the change of the tropism of the PIV vector and must prevent induced immunosuppression. for the measles virus. The lack of an effective vaccination against measles virus infection results in the death of more than 2,700 children worldwide on a worldwide basis. The HPIV3r-NBHA candidate vaccine (p-M) offers a unique opportunity to immunize against the two main causes of severe pediatric disease, namely, HPIV3 and measles viruses. Unlike currently authorized measles virus vaccines, we expect chimeric HPIV3r-NB HA (PM) and other human-bovine chimeric vector constructs, which express the major antigenic determinant of measles virus or other heterologous pathogens, to be used to induce an important immune response for, for example, measles virus, in infants and children younger than six months of age (Durbin, J. Virol. 74 ^ 6821-31,2000). An effective immunization strategy for infants and children will be used to address the goal of the World Health Organization to eradicate measles by 2010. In particular, a measles virus vaccine that does not include a virus would be advantageous for eradication. of infectious measles.

EXAMPLE X Use of bovine-human parainfluenza type 3 virus (B / H PIV3r) as a vector for supernumerary RSV glycoprotein genes For use within the present invention, a recombinant chimeric human-bovine PIV was constructed in which F and HN genes of BPIV3 were replaced with those of HPIV3. This recombinant chimeric human-bovine B / HPIV3r virus was shown to be fully competent for replication in cell culture, whereas in rhesus monkeys it exhibited the phenotype characteristic, restricted to the host classification of BPIV3 and was quite immunogenic and protective ( U.S. Patent Application Serial No. 09/586, 479, filed June 1, 2000 by Schmidt et al. / Schmidt et al., J. Virol. : 8922-9, 2000, each incorporated herein by reference). This is another example of a "modified Jennerian" proposal that is useful within the compositions and methods of the invention, but in this case the complete set of "internal" viral genes is derived from BPIV3, with the antigenic determinants alone derived from HPIV3. . As noted above, there are many practical and safe considerations that favor vaccines based on an individual PIV3 structure, as opposed to a complex mixture of different viruses each of which must be attenuated and verified separately and which can interact in unpredictable. In addition, the host classification restriction of BPIV3 confers an attenuation phenotype that must be very stable. In the present example, a recombinant chimeric human-bovine PIV3 (B / HPIV3r) coding for glycoprotein G or F of the respiratory syncytial virus (RSV), which are the main neutralizing and protection antigens of RSV, was designed, rescued and characterized. This example shows that the B / HPIV3r quickly accepted the foreign RSV genes without a significant reduction of its replicative efficiency in vitro or in vivo and thus is a promising candidate vector and vaccine. This vector will be free of poor development problems ?? Vitreous and instability that are otherwise characteristic of RSV.

Construction of antigenomic cDNAs encoding recombinant chimeric B / HPIV3 viruses carrying an ORF G or F of RSV subgroup A as an additional supernumerary gene A full length cDNA of BPIV3 strain Kansas was constructed in which F and HN glycoprotein genes of the bovine virus had to be replaced with the corresponding genes of the JS strain of HPIV3 (B / HPIV3r) (U.S. Patent Application Serial No. 09 / 586,479, filed on 1 June 2000 by Schmidt et al., Schmidt et al., J. Virol. 74: 8922-9, 2000, each incorporated herein by reference). For use within the present invention, this cDNA was modified to contain three additional single restriction enzyme recognition sites. Specifically, the BlpI site was introduced preceding the N ORF (nucleotide (nt) 103-109), an AscI site was introduced preceding the final N gene sequence and a Notl site was introduced preceding the final sequence of the P gene These restriction enzymatic recognition sites were introduced to facilitate the insertion of foreign supernumerary genes into the genome of the chimeric B / HPIV3 virus genome. The sites were designed in such a way that they did not break any of the cis-acting elements of replication and tanscription of BPIV3. this specific example will describe 1 insertion in the BlpI site (Figure 13). The G and F genes of the RSV subgroup A described above (Accession No. GenBank M74568) were modified for insertion into the nearby BlpI promoter site of B / HPIV3 (Figure 13). The strategy was to express each heterologous ORF as a separate, additional mRNA and therefore it was important that it be introduced into the B / HPIV3r genome in such a way that it was preceded by a BPIV3 gene start signal and followed by a final signal from BPIV3 gene. The BlpI insertion site followed the gene start signal of the N gene (Figure 13). Thus, for insertion in this site, the RESV ORF needed to be modified by the insertion of a BlpI site at its 5 'end and the addition of an end signal of BPIV3 gene, intergenic region, gene start signal, and BlpI site at its 3 'end. ORF AG for RSV, the PCR primer used was direct (5 'to 3') AATTCGCTTAGCGATGTCCAAAAACAAGGACCAACGCACCGC (SEQ ID NO.30), the reverse primer was (5 'to 3') AAAAAGCTAAGCGCTAGCCTTTAATCCTAAGTTTTTCTTACTTTTTTTACTAC TGGC GTGGTGTGTTGGGTGGAGATGAAGGTTGTGATGGG (SEQ ID NO. 31) (BlpI underlined site, initiation triplets and ORF translational ending in bold). ORF for AF RSV, direct PCR primer was used (5 'to 3') AAAGGCCTGCTTAGCAAAAAGCTAGCACAATGGAGTTGCTAATCCTCAAAGCA GCAATTACC AAT (SEQ ID NO.32), and the reverse primer was (5 'to 3') AAAAGCTAAGCGCTAGCTTCTTTAATCCTAAGTTTTTCTTACTTTTATTAGTT ACTAAATGCAA TTATTTATACCACTCAGTTGATC (SEQ ID .NO 33) (BlpI site underlined, initiation triplets and translational termination ORF in ne, cries.) PCR products were digested with BlpI and cloned into the modified full-length cDNA clone using standard molecular cloning techniques. The resulting full-length isolate containing the RSV ORF AG was designated pB / HPIV3-GA1 and the plasmid containing the ORF F was designated pB / HPIV3-FA1 The nucleotide sequence of each inserted gene was confirmed by restriction enzymatic digestion and automated sequencing.All constructs were designed in such a way that the nucleotide length of the final genome was a multiple of six, which al has been shown to be a requirement for efficient RNA replication (Calain et al., J. Virol. 67: 4822-30, 1993, incorporated herein by reference).

Recovery of the chimeric viruses B / HPIV3r-Gl and B / HPIV3r-Fl from the cDNA. The B / HPIV3r-Gl and B / HPIV3r-Fl viruses were recovered from the pB / HPIV3-GA1 and pB / HPIV3-FA1 cDNAs, respectively. This was carried out by the method described above in which the HEp-2 cells were transfected with the respective antigenomic cDNA with the supporting plasmids N, P and L of BPIV3. The cells were simultaneously infected with a recombinant vaccinia virus, strain MVA, which expresses the T7 RNA polymerization gene. The recovered recombinant viruses were biologically cloned by sequential terminal dilution in Vero cells. The presence of the RSV G or F gene inserted into the structure of each recovered recombinant virus was conformed by RT-PCR of the viral RNA isolated from the infected cells followed by enzymatic digestion by restriction and DNA sequencing. The sequence of the inserted gene and the flanking regions in the recovered recombinant viruses was identical to that of the starting antigenomic cDNA.

B / HPIV3r-Gl and B / HPIV3r-Fl viruses replicated in cell culture The multiclyclic growth kinetics of B / HPIV3r-Gl and B / HPIV3r-Fl in LLC-MK2 cells was determined by infecting MLC-CLL monolayers with triplicate in a multiplicity of infection (MOI) of 0.01 and harvest samples at 24 hour intervals with respect to a seven day period, as described above (Bailly et al., J.

Virol. 11: 3188-3195, 2000a, incorporated herein by reference). These two viruses were compared with Ka of BPIV3, JS of HPIV3, Ka of BIV3r, and B / HPIV3r (Figure 14). The two precursor viruses carrying HPIV3 glycoproteins, namely HPIV3 and B / HPIV3r, appeared to replicate somewhat more rapidly than the others. However, the final degree achieved for each of the six viruses was similar with one exception: B / HPIV3r-Fl was reduced approximately 8-fold in its replicative capacity compared to the other viruses (Figure 14). This could be an effect of having this large gene in a proximal-promoter position, or it could be an effect of the expression of a second fusogenic protein or both. This latter possibility was suggested by the observation that the B / HPIV3r-Fl induced syncytially in massive form was comparable with what was observed with wild-type RSV infection and higher than that observed with B / HPIV3r or the other precursor viruses. In comparison, B / HPIV3r-Gl induced less cytopathic and less syncytial effect in LLC-MK2 cells, comparable to B / HPIV3r. However, B / HPIV3r-Fl and B / HPIV3r-Gl grew to a final titer of at least 107 TCID50 / ml in LLC-MK2 cells and in Vero cells. This indicated that each virus is completely tolerant for growth which will result in an efficient cost of manufacturing the vaccine.

The virus of B / HPIV3r-Gl and B / HPIV3r-Fl replicated efficiently in the respiratory tract of hamster B / HPIV3r-Gl and B / HPIV3r-Fl were evaluated for their ability to replicate in the upper and lower respiratory tract of hamsters . The precursor virus of B / HPIV3r, as well as the biologically derived viruses BPIV3 and HPIV3 were compared in parallel as controls (Table 16). Each virus was administered intranasally at a dose of 106 TCID5o / -and one group received both B / HPIV3r-Gl and B / HPIV3r-Fl. The animals of each group were sacrificed at 4 and 5 days after infection and the virus titer in the nasal turbinates and lungs was determined by serial dilution. The level of replication of B / HPIV3r-Gl in the respiratory tract was very similar to that of JS of HPIV3 and Ka of BPIV3, and statistically indistinguishable from them. The replication of B / HPIV3r-Fl appeared to be reduced to some degree on days 4 and 5 in relation to the others, but this difference was not statistically significant in comparison with the biological BPIV3 virus, which in previous primate and clinical studies was replicated sufficiently well to induce an immunoprotective response (Coelingh et al., Virology 162; 137-143, 1988; Karron et al., Pediatr. Infect. Dis. J. 15: 650-654, 1996, each incorporated herein). as reference) .

Also, the virus titer from the mixed infection of B / HPIV3r-Gl and B / HPIV3r-Fl appeared to be reduced to some extent in the lower respiratory tract on day 4, but this was not statistically significant. The replication of one of the control viruses, Ka of BPIV3 Ka, was somewhat reduced in the lower respiratory tract on day 5; this was also not statistically significant and indicates that these small differences are probably not important. In this way, B / HPIV3r-Gl and B / HPIV3r-Fl viruses appeared to be fully competent for replication in vivo, despite the presence of the 0.9 kb G or 1.8 kb F supernumerary gene after the promoter.

Table 16 The B / HPIV3r that carries the QRF G or F of RSV as a 9n supernumerary in the proximal promoter position efficiently replicates in the respiratory tract of hamsters. Immunization virus8 Mean number of virus titre in the mean of the virus titre in animals day 4"(log10TCID60 / g ± SE) c 5b (logloTCID5o / g ± S. E) c Turbined Turbine lungs Nasal nasal lungs B / HPIV3r-Gl 6 5.9 ± 0.1 (AB) 5.1 ± 0.6 (A) 5.5 + 0.2 (A) 5.6 ± 0. (AC) B / HPIV3r-Fl 6 5.1 ± 0.3 (B) 4.6 ± 0.2 (A) 5.7 ± 0.2 (AB) 3.6 ± 0.2 (BD) B / HIV3r-Gl & B / HPIV3-F1 6 5.7 ± 0.3 (BC) 4.3 ± 0.8 (A) 5.6 ± 0.2 (A) 5.9 ± 0.2 (A) B / H IV3 6 6.2 ± 0.2 (AC) 5.2 ± 0.6 (A) 6.5 ± 0.1 (B) 5.7 ± 0.6 (AC) JS of HPIV3 wild type 6 6.6 ± 0.1 (A) 6.5 ± 0.1 (A) 6.0 ± 0.2 (AB) 6.0 ± 0.4 (A) Ka of BPIV3 wild type 6 5.8 ± 0.1 (AB) 6.1 ± 0.2 (A) 5.3 ± 0.2 (A) 4.2 ± 0.5 < CD) The hamsters were inoculated intranasally with 10eTCID50 of the virus in an inoculum of 0.1 ml. The animals were sacrificed on day 4 or 5 after inoculation, as indicated, and virus titers in the nasal turbinates and lungs were determined by titration in LLC-MK2 (PIV3) or HEp-2 (RSV) cells at 32 * C. The limit of detectability of the virus was 102.45TCID5o / g of tissue. HE = standard error. The means of the virus titers were assigned to similar groups (A, B, C, D) by the Tukey-Kramer test. Within each column, the average titles with different letters are statistically different (p <0.05). The titles indicated with two letters are not significantly different from those indicated with any letter.

B / HPIV3r-Gl and B / HPIV3r-Fl viruses induce serum antibodies for both HPIV3 and RSV Hamsters were infected with B / HPIV3r-Gl, B / HPIV3r-Fl or B / HPIV3r as described above. One additional group received both B / HPIV3r-Gl and B / HPIV3r-Fl and another group was infected intranasally with RSV. Serum samples were collected 5 days after infection and analyzed for RSV-specific antibodies by an ELISA test specific for the RSV F protein or the RSV G protein (Table 17), and for HPV3 HN specific antibodies by the analysis of antibody to inhibit haemagglutination (HAI) (Table 18). The titer of antibodies specific for F or specific for G induced by virus B / HPIV3r-Fl or B / HPIV3r-Gl, respectively, was 2 to 4 times higher than that induced by wild-type RSV. Animals inoculated with both B / HPIV3r-Fl and B / HPIV3r-Gl also had high specific F and specific G antibody titers. In addition to the high ELISA titers against GV and F of RSV, B / HPIV3r-Gl and B / HPIV3r-Fl also induced neutralizing serum antibody titers for RSV that were superior to those induced by wild-type RSV (Table 18). Each of the viruses induced a specific antibody titer for PIV3 that could not be distinguished from that of its precursor virus B / HPIV3r (Table 18). In this way, vector B / HPIV3r carrying the F or G gene of RSV induced important immune responses both against the RSV insert and against the PIV vector.

Table 17 Immunization of hamsters with B / HPIV3r expressing the RSV G or F ORF as a supernumerary gene induces an antibody response against the G protein or RSV virus. 8 Immunization Animals 8 Title ELISA Serum IgG ELISA IgG serum against RSV6 protein F RSV5 protein G group (mean reciprocal (mean reciprocal log2 ± SE ^) log; ± SE) e Day 26 Pre-Day Increase 26 Increase log2 ~ times log2-fold B / HPIV3r-Gl 12 6.0 ± 0.4 = 12.5 ± 0.5 6.5 6.7 ± 0.5C. 7.5 ± 0.5 0.8 B / HPIV3r-Fl 12 6.3 ± 0.3 7.2 ± 0.3 0.9 6.8 ± 0.3 16.2 ± 0.5 9.4 B / HPIV3r-Gl & B / HPIV3r-Fl? 12 6.5 ± 0.6 12.0 ± 0.9 5.5 7.310.5 14.7 ± 0.4 7.4 I heard B / HPIV3r 12 6.5 ± 0.4 8.0 ± 0.4 1.5 7.3 ± 0.7 8.3 ± 0.8 1.0 RSV 12 6.8 ± 0.3 10.8 ± 0.4 4.0 7.3 ± 0.5 15.7 ± 0.4 8.2 The hamsters were inoculated intranasally with 106 TCID¾0 of the virus in an inoculum of 0.1 ml. Serum samples were extracted on day 26 after inoculation and analyzed by glycoprotein-specific ELISA for antibodies against the G or F protein of FSV, as indicated. The titres in the pre-serum specimen represent non-specific background levels of antibody in this sensitive ELISA assay.

Table 18 Immunization of hamsters with B / HPIV3r expressing the ORF G or F of RSV that induces neutralizing antibodies against RSV as well as antibodies to inhibit haemagglutination (HAI) against HPIV3.

Immunization virus * Animals Antibody response HAI antibody response per serum neutralizing group for serum RSV * for HPIV3C (mean (reciprocal mean log2 ± SE) d reciprocal log2 ± SE) d re Day 26 Pre Day 26 B / HPIV3r-|Gl 12 = 3.3 10.0 ± 0.3 (A) = 2 10.0 ± 0.5 (A) B / HPIV3r-|Fl 12 = 3.3 9.3 ± 0.5 (A) = 2 8.8 ± 0.1 (A) B / HPIV3r- • Gl £ B / HPIV3r -Fl 12 = 3.3 10.8 ± O.4 (A) = 2 8.8 ± 0.3 (A) B / HPIV3r 12 = 3.3 0.810.8 (B) = 2 9.5 ± 0.8 (A) RSV 12 = 3.3 8.1 ± 1.2 (A ) = 2 = 2 < B) The hamsters were inoculated intranasally with 106 TCID50 of the indicated PIV3 or with the PFU of RSV in an inoculum of 0.1 ml. Serum samples were extracted on day 26 after inoculation and antibody titers were determined by neutralization test by 60% plate reduction. Serum samples were extracted on day 26 after inoculation and antibody titers were determined by tests for hemagglutination inhibition. The means of the virus titers were assigned to similar groups (A, B) by the Tukey-Kramer test. Within each column, the average titles with different letters are statistically different (p <0.05).

B / HPIV3r-Gl and B / HPIV3r-Fl viruses induce resistance to replication of the inoculated HPIV3 and RSV virus Hamsters immunized with B / HPIV3r, B / HPIV3r-Gl, B / HPIV3r-Fl or B / HPIV3r-Gl plus candidates for B / HPIV3r-Fl vaccine were inoculated 28 days later by intranasal inoculation of 106 TCID50 of HPIV3 or 106 PFU of RSV. The animals were sacrificed 5 days later and the nasal turbinates and lungs were harvested and virus titers were determined (Table 19). The animals that had received the precursor virus B / HPIV3r or the Gl and Fl derivatives exhibited a high level of resistance to the replication of the HPIV3 inoculated virus and there were no significant differences between the experimental groups. The animals that received B / HPIV3r-Gl, or B / HPIV3r-Fl, or the two viruses, exhibited a high level of resistance to replication of the inoculated RSV virus. The level of protective efficacy of the B / HPIV3r-Fl virus against the inoculated RSV appeared to be marginally lower than that of the B / HPIV3r-Gl virus or the control RSV. However, this difference was not significantly different. Thus, vector B / HPIV3r carrying either the F or G gene of RSV induced a level of protective efficacy that was comparable to that of complete infectious RSV.

Table 19 Immunization of hamsters with B / HPIV3r-Gl and / or B / HPIV3r-Fl induces resistance to inoculation with HPIV3 and RSV 28 days after infection. Immunization virus * Not from HPIV3b Mediates Title average RSV0 Animals (log0TCI50 / g ± SE) d (log10UFP / g ± SE) 'Turbinados lungs Turbinados nasal nasal lungs B / HPIV3r-Gl 6 2.3 ± 0.1 (A) 3.1 ± G .2 (A) 1.9 ± 0.2 (AB) = 1.7 (A) B / HPIV3r-Fl 6 2.6 ± 0.2 (A) 3.1 ± 0.1 (A) 2.9 ± 0.4 (BC) 2.1 ± 0.2 (A) B / HPIV3r-Gl & B / HPIV3r-Fl 6 2.8 ± 0.2 (A) 2.8 ± 0.3 (A) 1.8 ± 0.1 (A) 1.9 ± 0. (A) B / HPIV3r 6 2.3 ± 0.5 (A) 3.6 ± 0.4 (A) 4.1 ± 0.5 (C) 3.5 ± 0.4 (B) RSV 6 5.6 ± 0.2 (A) 5.2 ± 0.2 (B) 1.9 ± 0.3 (AB) ) = 1.7 <A) Groups of 6 hamsters were inoculated intranasally with 106 TCID50 of the indicated PIV3 or with 106 PFU of RSV in an inoculum of 0.1 ml. The HPIV3 titers were performed on LLC-MK2 cells. The limit of detectability of the virus was 101-7TCID50 / g of tissue. The quantification of RSV was determined by plate numbering on HEp-2 cells. The limit of detectability of the virus was 101'1 PFU / g of tissue. The means of virus titers were assigned to similar groups (A, B, C) by the Tukey-Krame test. Within each column, the average titles with different letters were statistically different (p < 0.05). The titles indicated with two letters are not significantly different from those indicated with any letter.

EXAMPLE XI Use of B / HPIV3r.l as a vector for the HN and F protein of hemagglutinin of PIV2 The HPIV3r-l chimeric virus, which had an HPIV3 structure in which the HPIV3 HN and F genes have been replaced by their counterparts HPIV1, serves as a useful vector for the HN protein of HPIV2 as a supernumerary gene. This chimeric vector, HPIV3rl.2HN, is demonstrated herein to induce replication resistance of both HPIV1 and HPIV2 in hamsters. These findings illustrate the surprising flexibility of the PIV expression system. For example, the HPIV3r-1.2HN recombinant virus contains elements of each of the three serotypes of HPIV that cause significant disease: the internal serotype 3 genes combined with the glycoprotein HN and F genes of serotype 1 and the protective antigen HN of serotype 2 as a supernumerary gene. The present example still provides another proposal for deriving a vector vaccine based on PIV to protect against both PIV1 and PIV2. In this example, the B / HPIV3r was modified by replacing the human PIV3 HN and F proteins with those of HPIV1. This virus, designated B / HPIV3r.l, contains the HN and F glycoproteins of PIV1 as part of the vector structure, intended to induce neutralizing antibodies and immunity to HPIV1. This virus was used in the present example as a vector for expressing the HN and F proteins of HPIV2 individually or together as supernumerary genes. Three viruses were recovered and showed to be completely viable: B / HPlV3r .1-2F; B / HPIV3r .1-2HN; or B / HPIV3r .1-2 F, 2HN, and each expressed the F and / or HN gene of PIV2 as a supernumerary gene or genes. B / HPIV3r .1-2F, 2HN, which expressed both the F and / or HN proteins of PIV2, from two supernumerary genes and the F and HN genes of PIV1 from the vector structure, thus expressing the two protective antigens principal, that is, the F and HN of glycoproteins, of PIV1 and PIV2 from an individual virus. This proposal optimizes the protective efficacy of the vaccine and minimizes manufacturing costs since it carries out this enhanced immunogenicity using only one virus. It will also probably be simpler, safer and more effective to immunize infants and children with a single multivalent virus compared to a mixture of several viruses.

Construction of the antigenomic cDNAs encoding the recombinant chimeric B / HPIV3r.l viruses carrying the FIV and HN genes of HPIV2 as additional supernumerary genes A full-length cDNA of the BPIV3 strain ansas in which the glycoprotein F genes and HN of the bovine virus has been replaced with the corresponding genes of the JS strain of HPIV3 (B / HPIV3r) was constructed as described above (Schmidt et al., J. Virol. 74: 8922-9, 2000, incorporated herein) as reference) . This cDNA was modified to contain three additional single restriction enzyme recognition sites (Figure 15). Specifically, the BlpI site was introduced preceding the N ORF (nucleotide (nt) 103-109), an AscI site (nt 1676-83) was introduced preceding the N gene end sequence and an iVotl site (nt 3674-3681). ) was introduced preceding the final sequence of the P gene. Then, the glycoprotein F and HN genes of B / HPIV3r were replaced with the corresponding HPIV1 genes. To accomplish this, subclone 3.1hcR6 of the full length cDNA of HPIV3r-l described above (Tao et al., J. Virol.72: 2955-2961, 1998, incorporated herein by reference), which contained the ORFs of the glycoprotein F and HN genes of HPIV1 under the control of the HPIV3 transcription signals were modified by PCR mutagenesis to create an SgrAI restriction enzyme recognition site preceding the F gene and a BsiWI site preceding the final sequence of the HN gene , analogous to the position of the SgrAI and BsiWI sites that had previously been introduced in rB / HPN3 (Schmidt et al., J. Virol. 74: 8922-9, 2000). The mutagenic direct primer used to create the SgrAI site was (5 'to 3') CGGCCGTGACGCGTCTCCGCACCGGTGTATTAAGCCGAAGCAAA (SEQ ID NO: 34) (underlined SgrAI site), and the mutagenic reverse primer was (5 'to 3) CCCGAGCACGCTTTCTCTTAGTAAG TTTTTATATTTCCCGTACGTCTATTGTCT GATTGC (SEQ ID NO.35) (Bsi I site underlined). The grAI and BsiWI sites were used to replace, as a single DNA fragment, the FN and HN genes of HPN3 in B / HPIV3 with the F and HN genes of HPIV1 from the modified 3.1hcR6 plasmid. This provided the full length antigenomic cDNA pB / HPIV3.1, which consists of F and HN open reading frames of HPIV1 in accordance with the control of the HPIV3 transcription signals in an antecedent that is derived from BPIV3. In the next step, the F and HN open reading frames of HPIV2 described above (access numbers GenBank AF213351 and AF213352) were modified for insertion into the Notl and AscI sites, respectively, of pB / HPN3.1 (Figure 15) . The strategy was to express each ORF and HN of PIV2 as an additional separate RNAiri and therefore it was important that it be introduced into the B / HPIV3r genome in such a way that it was preceded by a PIV3 gene start signal and followed by the end signal of PIV3 gene. The Notl insertion site precedes the gene end signal of the P gene (Figure 15). Therefore, for the insertion in this site, the ORF F of HPIV2 needed to be modified for the insertion of a. Wotl site and the addition of an end-of-gene signal of BPIV3, intergenic region and gene start signal at its 5 'end and a Wotl site at its 3' end. ORF F for HPIV2, the PCR primer used was direct (5 'to 3') AAAATATAGCGGCCGCAAGTAAGAAAAACTTAGGATTAAAGGCGGATGGATCA CCTGCATCCAATGATAGTATGCATTTTTGTTATGTACACTGG (SEQ ID NO.36) and the reverse primer was (5 'to 3') AAAATATAGCGGCCGCTTTTACTAAGATATCCCATATATGTTTCCATGATTGT TCTTGGAAAAGACGGCAGG (SEQ ID NO. 37) Wotl site underlined) ORT translational initiation and completion triplets in bold). For the HPIV2 HN ORF, the same cis-acting elements were added as described above for HPIV2 F, but instead of Wotl, an AscI site was added on either side of the insert to facilitate cloning at the NP gene junction. . The direct PCR primer used was (5 'to 3') GGAAAGGCGCGCCAAAGTAAGAAAAACTTAGGATTAAAGGCGGATGGAAGATT ACAGCAATCTATCTCTTAAATCAATTCC (SEQ ID NO: 38), the reverse primer was (5r to 3 ') GGAAAGGCGCGCCAAAATTAAAGCATTAGTTCCCTTAAAAATGGTATTATTTG G (SEQ ID NO 39). The PCR products were digested with Wotl (insert F of HPIV2) or Ase! (HN insert of HPIV2) and cloned into the modified full-length cDNA clone using standard molecular cloning techniques. The resulting full-length cDNA containing the ORF F of HPIV2 was designated pB / HPIV3.1-2F, the full-length cDNA containing the ORF HN of HPIV2 was designated pB / HPIV3.1-2HN and the plasmid containing the Both F and HN inserts were designated pB / HPIV3.1-2F, 2HN. The nucleotide sequence of each inserted gene was confirmed by restriction enzymatic digestion and automated sequencing. All constructs were designed in such a way that the final genomic nucleotide length was a multiple of six, which has been shown to be a requirement for efficient RNA replication (Calain et al., J. Virol. 67: 4822-30 , 1993, incorporated herein by reference). The genomic nucleotide length of the recovered chimeric viruses is as follows: pB / HPIV3.1: 15492; pB / HPIV3.1-2HN: 17250; pB / HPIV3.1-2F: 17190; PB / HPIV3.1-2-2HN, 2F: 18948.

Recovery of chimeric viruses B / HPIV3r.l, B / HPIV3r .1-2F, B / HPIV3r .1-2HN and B / HPIV3r .1-2F, 2HN from cDNA Chimeric viruses B / HPIV3r.l, B / HPIV3r.l-2F, B / HPIV3r .1-2HN and B / HPIV3r .1-2F, 2HN; were recovered from cDNAs pB / HPIV3.1, pB / HPIV3.1-2F, PB / HPIV3.1-2HN and pB / HPIV3.1-2F, 2HN respectively. This was carried out by the method described above in which the HEp-2 cells were transfected with the respective antigenomic cDNA together with the support plasmids N, P and L of BPIV3. The cells were simultaneously infected with a recombinant vaccinia virus, strain MVA, which expresses the T7 RNA polymerase gene. Porcine trypsin was added to the cell culture medium to activate the F protein of HPIV1, as described previously (Tao et al., J. Virol. 72: 2955-2961, 1998). The recovered recombinant viruses were biologically cloned by sequential terminal dilution in Vero cells. All recombinant viruses replicated efficiently, induced CPE in Vero cells within 5 days and made the cell monolayer positive for hemadsorption. The presence of the F and HN gene of HPIV2 inserted into the structure of each recovered recombinant virus was confirmed by RT-PCR of viral RNA isolated from infected cells followed by enzymatic restriction digestion and DNA sequencing. The sequence of the inserted gene and flanked regions in the recovered recombinant viruses was identical to that of the starting antigenomic cDNA.

EXAMPLE XII Use of HPIV3r-l cp45L as a vector for the hemagglutinin protein (HA) of the measles virus: development of a sequential immunization strategy The HPIV3r-l chimeric virus, which had an HPIV3 structure in which the HN genes and F of HPIV3 have been replaced by their counterparts HPIV1, shown above to serve as a useful vector for the HN protein of HPIV2 as a supernumerary gene. This chimeric vector, HPIV3r-l .2HN, was able to induce replication resistance of both HPIV1 and HPIV2 in hamsters. This finding illustrates the surprising flexibility of the PIV expression system. For example, this particular virus, HPIV3r-l .2HN, contained elements for each of the three serotypes of HPIV: the internal genes of serotype 3 combined with the glycoprotein genes HN and F of serotype 1, and the protective antigen HN of the serotype 2 as a supernumerary gene. An additional derivative, HPIV3r-1.2HNcp45L, was also made to contain attenuating mutations of the candidate for cp45 HPIV3 vaccine. In this way, a PIV vector may be represented to comprise three components: the internal vector structure genes, which may contain attenuation mutations as desired; the vector glycoprotein genes, which may be of the same serotype or a heterologous serotype; and one or more supernumerary genes that code for protective antigens for additional pathogens. In most cases, these supernumerary antigens are not incorporated into the virion and therefore do not change the neutralization or tropism characteristics of the virus. In this way, each PIV vector is a bivalent or multivalent vaccine in which the vector itself induces immunity against a major human pathogen and each supernumerary antigen induces immunity against an additional pathogen. In the present example, the flexibility of the PIV vector system is further demonstrated by using HPIV3r-l virus, as well as its attenuated HPIV3r-lcp45L derivative, as vectors for expressing Ha of measles virus as a supernumerary gene. This provides a new candidate for bivalent vaccine for the HPIV1 virus and measles. In this way, the HA of the measles virus can be vectorized by HPIV3r and attenuated derivatives thereof, which carry the antigenic determinants of serotype 3, or by HPIV3r-l and attenuated derivatives thereof, which carry the antigenic determinants of serotype 1. It is It is important to mention that the three serotypes of HPIV (1, 2 and 3) do not confer significant cross protection and that each represents a significant human pathogen for which a vaccine is needed. This gives rise to the possibility that three serotypes could be used to sequentially immunize the infant against the IVP as well as protective antigens vectorized against heterologous pathogens. Specifically, immunization with a PIV vector containing the antigenic determinants of a serotype must be minimally or in no way accepted by the above immunization with a vector or vectors containing the antigenic determinants of a heterologous serotype. This provides the opportunity to perform sequential immunizations and boosts (preferably at intervals of 4-6 weeks or longer) against supernumerary antigens, as well as, against the three HPIV serotypes, whose genes can be expressed either in the vector structure or as supernumerary genes. The present example details the use of reverse genetics techniques to develop a candidate vaccine of HPIV1 attenuated in vivo, PlV3r-lHAp-.Mcp 5L, which expresses as a supernumerary gene the protective antigen of the major measles virus, the HA glycoprotein ( Durbin, J. Virol. 74: 6821-31, 2000, incorporated herein by reference), for use in infants and younger children to induce an immune response both against the measles virus and against HPIV1. Also, a sequential immunization scheme was developed in which immunization with the HPIV3r candidate vaccine attenuated HAP_M cp45L (which carries the antigenic determinants of serotype 3) was followed by the candidate vaccine HPIV3r-l HAP-M cp45L (which carries the antigenic determinants of serotype 1). The hamsters immunized with these viruses developed antibodies for the antigens - HPIV3 and HPIV1 present in the structure of the vectors and also maintained high titres of antibodies for the vectorized antigen, the HA of the measles virus expressed as a supernumerary antigen from the viruses for vaccine candidates for both HPIV3 and HPIV1.

Construction of the wild type and attenuated versions of HPIV3r-l HA (PM) and HPIV3r-l HA (PM, cp45L, of HPIV3r-l expressing measles virus HA as a supernumerary gene) Two full-length plasmids, pFLC, were constructed HPIV3-1 HA (p_M) and pFLC HPIV3-1 HA, P_M) cp45L (Figure 16) as described above (see also, Durbin, J. Virol. 74: 6821-31, 2000; Skiadopoulos et al., J. Virol., 72: 1762-8, 1998; Tao et al., J. Virol. 72: 2955-2961, 1998, each incorporated herein by reference). pFLC HPIV3-1 HA (pM) was constructed using the pFLC HPIV3 HA (PM) described above in which the ORF of the HA gene of the Edraonston strain of wild type measles virus was inserted as a supernumerary gene between the P and M genes of HPIV3r. pFLC HPIV3HA (P-M) was digested with BspEI to Sphl and the cDNA fragment lacking the sequence BspEI to Sphl of 6487 base pairs was isolated. Then, pFLC 2G + .hc, a full length antigenomic cDNA plasmid carrying the F and HN ORFs of PIV1 in place of those of HPIV3 (Tao et al., J. Virol 72; 2955-2961, 1998) was digested with BspEI and Sphl and the fragment of 6541 base pairs (plasmid nts 4830-11371) containing the glycoprotein genes HPIVl in the HPIV3 structure was inserted into the BspEI window to Sphl of pFLC HPIV3 HAP_M to provide pFLC HPIV3-1 HAP- M (Figure 15). The cp45 L mutations present in the ORF of the L gene (point mutations coding for the amino acid substitutions Ser-942 to His, Leu-992 for Phe and Thr-1558 for lie) are the main determinants ts and att of the candidate vaccine of HPIV3 cp45 (Skiadopoulos et al., J. Virol. 72: 1762-8, 1998) and were previously shown to co replication attenuation for HPIV3r-l cp45L in the hamster respiratory tract (Tao et al., Vaccine 17: 1100-8, 1999). PFLC HPIV3-1 HAP-M was then modified to encode these three ts mutations to provide pFLC HPIV3-1 HAP_M cp45L (Figure 16). This was carried out by inserting the restriction endonuclease fragment Sphl to NgoMIV from pFLC HPIV3 cp45L (plasmid nts 11317 -15929) (Skiadopoulos et al., J. Virol. 72: 1762-8, 1998) in the Sphl window to NgoMIV of pFLC HPIV3-1 HAP-M.

Recovery of HPIV3r-l HA (PM> and HPIV3r-l HA (PM) cp45L pFLC HPIV3-1 HA (PM) or pFLC HPIV3 * 1 HA (PM) cp45L was transfected separately into HEp-2 cells in plates of six cavities (Costar, Cambridge, MA) together with plasmids support pTM (N), pTM (P no C) and pTM (L) and LipofectACE (Life Technologies, Gaithersburg, MD) and the cells were simultaneously ited with MVA-T7, a r4ecombinant for vaccinia virus with defective replication that codes for bacteriophage T7 polymerase protein as described previously (Skiadopoulos et al., Vaccine 18: 503-10, 1999b, incorporated herein by reference). After incubation at 32 ° C for four days in medium containing trypsin, the transfection collection was passed over LLC-MK2 cells in a 25 cm2 flask and the cells were incubated for 5 days at 32 ° C. Virus recovered from the cell supernatant was further passaged on LLC-MK2 monolayer cultures with trypsin at 32 ° C to amplify the virus. PIV3r-l HAP.M and PIV3r-l HAP- "cp45L were biologically cloned by terminal dilution in LLC-MK2 monolayer cultures at 32 ° C as described previously (Skiadopoulos et al., Vaccine 18: 503-10,1999b). Viral suspensions derived from the biologically cloned virus were amplified on LLC-MK2 monolayer cultures. Viral RNA (vRNA) was isolated from biologically cloned recombinant chimeric viruses as described above. RT-PCR was performed using HPIV3r-l HAP_M or HPIV3r-l HAP-M cp45L vRNA as a template and specific oligo-nucleotide primers that extended the HA gene insert or the cp45 mutations in the L gene. The RT-PCR products were analyzed by restriction endonuclease digestion and partial DNA sequencing of the PCR products as described above. This confirmed the presence of the HA gene of the measles virus inserted between the P and M genes of HPIV3r-1 and the presence of the mutations of the L-gene of cp45 in its attenuated derivative.

Demonstration of the attenuation phenotype of HPIV3r-l HA (EM) cp 5L in hamsters Golden Syrian hamsters in groups of 6 were inoculated intranasally with 106TCIDso of HPIV3r-l, HPIV3r-l HAP-M, HPIV3r-l cp45L or HPIV3r-l HAP-M cp45L. Four days after the inoculation, the nasal lungs and turbinates were harvested and the virus titers were determined as described previously (Skiadopoulos et al., Vaccine 18; 503-10, 1999b). The titles were expressed as the logico TCIP50 average / gram of tissue (Table 20). The recombinant HPIV3r-l HAP_M and its HPIV3r-1 -type precursor were replicated at comparable levels, indicating that the insertion of an additional transcription unit encoding the ORF of the HA gene does not further attenuate this hamster virus. The HPIV3r-l HAP ~ M cp45L and its HPIV3r-l cp45L precursor replicated at similar levels in the upper and lower respiratory tract indicating that HPIV3r-l HAP-M cp45L was successfully attenuated for replication in hamsters and that insertion of the ORF of the HA gene of the measles virus did not additionally attenuate the precursor virus of chimeric HPIV3r-l cp45L.

Table 2-0 Replication of the type and attenuated versions of the Ha virus of PIV3ir-1 and P! V3r-l in the respiratory tract of hamsters Vi us * Mean of the viri-s titre "(log1 (> TCID5o g ± SE) in: Nasal Turbinates Lungs t? TV3r-l type 6.3 ± 0; .1 6.6ft0.2 PIV3r-lHA '6.0 ± 0..1 5.740.7 PIV3r-l cp45L 4.1 ± 0.2 1.840. 2 PIV3r-lHA qp45L 4. ± 0.2 1.9Í0.2 Each of the -groups of 6 hamsters was inoculated with 106 TCIDso of the indicated virus intranasally. G The lungs and nasal turbinates were collected four days later. The virus presented in tissue homogenates was titrated by means of dilution in serum on L1C-MK2 memopy cultures at 32 ° C. Guinea pig erythrocytes were used for hemadsorption.

A specific immunization scheme which employs immunization with him candidate for chimeric HPIV3r attenuated HAp_Mcip45L vaccine, followed by he candidate for attenuated HPIV3r-l HAP-M cp45L vaccine induces antibodies for the HPIV3 and HPIV1 añtigéñós of vector structures and induces and he maintains high titers of antiseptic for him shared vectorized, the HA of the measles virus The infection of a hamster virus with HPIV3f-l HAP, M ep45L iñdüjó A response iñmuñé impófñáñté táñtó for him virus of HPIV1 as for him of measles (Table 21, group €) which indicates that HPIV3f-l, similar to HPIV3r could be a vector for him HA of the v rüs dé sáímpióñ. The feasibility of sequential immunization of hamsters with HPIV3r HAp-.Mcp45L and HPIV3r-l HAp-Mcp45L was then epilated. The groups of hamsters were immunized with 106 T £ lD5Q of HPIV3r HAp-MGp 5L (Table 21, groups 1, 2 and 3), HPIV3r cp45L (group 4), or control of measured L15 (group 5) (Table 21). 59 days after the first immunization, the groups of hamsters were immunized with 106 TCID50 of HPIV3r-l HAP_M ep45L (group 1 and 4), HPIV3f-l < 5p45L (group 2 and 5), or coding medium L15 (group 3). The serum methods were collected before the first immunization, 58 days after the first immunization and 35 days after the first immunization. Aimimals immunized with HPIV3r cp45B (Table 21, group 4) developed an important antibody response to HPIV3, and animals immunized with HPIV3r HAP_M cp45L (groups 1, 2 and 3) developed an important antibody response for both virus HPIV3 as measles. The animals in group 4, which had previously been immunized with HPIV3r cp45L / - were subsequently immunized with HPIV3r-l HAP.M cp45i, on day 59. When analyzed on day 94, these animals had high antibody titers against the HPIV3 virus and measles and a low to moderate level of antibodies to HPIV1. This showed that the virus for HPIV3-1 chimeric vaccine was capable of inducing an immune response for both the HPIV1 antigens of the vector and the HA protein vectorized even in the presence of immunity to HPIV3 but there was some decrease in this immunogenicity in immune animals. for HPIV3. The HPIV3r-l HAP-M cp45L vaccine was clearly immunogenic in animals previously immune to HPIV3 as indicated by the response of hamsters in group 4. These animals, which were immunized with HPIV3r cp45L on day 0, developed a moderately high titre of neutralizing antibodies to the measles virus on day 94, 35 days after immunization with HPIV3r-l HAp_M cp45L on day 59. Significantly , hamsters that were first immunized with HPIV3r HAP-M cp45L and then immunized with HPIV3r-l HAP.M cp45L (Group 1, Table 21) achieved a serum neutralizing antibody titre of higher measles virus on day 94 than groups of hamsters that were immunized with HPIV3r HAP-M cp45L (Group 3), suggesting that HPIV3r-l HAP-M cp45L can be used to maintain high levels of serum neutralizing antibodies for measles after immunization with HPlV3r HAP- M cp45L. Because the hamsters in group 1 developed this high antibody titer for measles virus HA after the first immunization with HPIV3r HAP-M cp45L, it was not possible to detect a four-fold or greater emergence of these titers after Immunization with HPIV3r-l HAP_M cp45L. In humans, it is likely that an HPIV3 vaccine such as, for example, HPIV3r HAP_M cp45L will be administered within the first four months of life followed two months later by a HPIV1 vaccine such as for example HPIV3r-l HAP-M cp45L ( Skiadopoulos et al., Vaccine 18: 503-10, 1999b, incorporated herein by reference). In contrast to rodents, human infants characteristically developed low antibody titers of viral glycoprotein antigens administered within the first six months of life, due to immunological immaturity, immunosuppression and maternal antibodies and other factors (Karron et al., Pediatr. Infect. Dis. J. 14: 10-6, 1995a; Karron et al., J. Infect. Dis. 172: 1445-1450, 1995b; Murphy et al. , J. Clin. Microbiol 2__: 894-8, 1986, each incorporated herein by reference). Therefore, it is very likely that a reinforcement effect of PIV3r-l HAP-M cp45L on the antibody titers for the measles virus HA will be necessary and will already be observed in those infants immunized with PIV3r HAP_M cp45L within the first six months of life. The present example indicates that it is possible to sequentially immunize animals with two attenuated PIV vaccines in vivo, serologically distinct, each of which expresses the measles virus HA, to develop antibodies to the HPIV3 and HPIV1 antigens of the vector structure and to maintain high titers of antibodies for the vectorized antigen, the HA of the measles virus.

Table 21 Sequential immunization of hamsters with PIV3r HA1P-M) cp45j, followed by PIV3r-l HA (pM) c 45L induces immunity for three viruses, namely HPIV1, HPIV3 and measles virus and maintains the antibody titre of the virus from saram ion to high levels to. The sera were collected 5 days before and 58 days after the first immunization. The second immunization was administered 59 days after the first one, and the serum was collected again 35 days later (day 94). b. The average HAI antibody titer of serum PIV3 is expressed as the reciprocal mean log2 1 standard error, SE. c. The mean of the serum neutralizing antibody titer for HPIV1 is expressed as the reciprocal mean log2 1 SE. d. The mean of the serum neutralizing antibody titer for the wild-type measles virus is expressed as the reciprocal mean log2 1 standard error, PRN, plaque reduction neutralization.

EXAMPLE XIII Construction and characterization of recombinants for chimeric HPIV3-2 vaccine expressing chimeric glycoproteins The present example details the development of a virus for PIV2 candidate vaccine attenuated in vivo for use in infants and young children using reverse genetics techniques. Preliminary efforts to recover the recombinant chimeric PIV3-PIV2 virus that carries the full-length PIV2 glycoproteins in a wild type PIV3 structure, as described above for the HPIV3-1 chimeric constructs, did not provide an infectious virus. However / viable PIV2-PIV3 chimeric viruses were recovered when the chimeric HN and F ORFs were used instead of the full-length PIV2 ORFs to construct the full-length cDNA. The recovered viruses, designated PIV3r-2CT in which the PIV2 ectodomain and the transmembrane domain were fused to the cytoplasmic domain PIV3 and PIV3r-2TM in which the PIV2 ectodomain was fused for the transmenbrane and cytoplasmic tail domain of PIV3, possessed similar phenotypes , although not identical in vitro and in vivo. Thus, it seems that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for a successful recovery of the PIV2-PIV3 chimeric viruses.

The recombinant chimeric viruses of PIV3r-2 exhibited an important host classification phenotype, i.e. they replicated efficiently in vitro although they were strongly restricted in in vivo replication. This attenuation in vivo occurs in the absence of any aggregated mutations of cp45. Although PIV3r-2CT and PIV3r-2TM were efficiently replicated in vitro, they were rather attenuated in the upper and lower respiratory tracts of hamsters and African green monkeys (AGMs), indicating that the chimerization of the HN and F proteins of PIV2 and PIV3 themselves specify an in vivo attenuation phenotype. A phenotype that includes efficient in vitro replication and highly restricted growth in vivo is desired in an important way for vaccine candidates. In spite of this attenuation, they were quite immunogenic and protective against inoculation with the wild-type PIV2 virus in both species. PIV3r-2CT and PIV3r-2TM were further modified by introducing the 12 PIV3 cp45 mutations located outside the HN and F coding sequences to derive PIV3r-2CTcp45 and PIV3r-2TMcp45. These derivatives were replicated efficiently in vitro although they were even further attenuated in hamsters and AGM indicating that the specific attenuation by glycoprotein chimerization and by cp45 mutations was additive. These findings identify the PIV3r-2CT and PIV3r-2TM recombinants as preferred candidates for use in the attenuated PIV2 vaccines in vivo.

Viruses and cells The wild type PIVI strain used in this study, PIVl / Washington / 20993/1964 (PIVl / wash64) (Murphy et al., Infect. Immun. 12: 62-68, 1975, incorporated herein by reference), was propagated in LLC-MK2 cells (ATCC CCL 7.1) as described previously (Tao et al., J. Virol. 72: 2955-2961, 1998 , incorporated herein by reference). The wild type virus of PIV, strain V9412-6, designated PIV2N94, was isolated in Vero cells qualified from a nasal lavage of a sick child in 1994. PIV2 / V94 was plaque purified three times on Vero cells before amplify twice in Vero cells using OptiMEM without FBS. The recombinant PIV3 / JS strain derived from wild type cDNA (PIV3r / JS) was propagated as described previously (Durbin et al., Virology 235: 323-332, 1997, incorporated herein by reference). The modified Vaccinia Ankara (MVA) recombinant expressing bacteriophage T7 RNA polymerase was generously provided by Drs. L. yatt and B. Moss (Wyatt et al., Virology 210: 202-205, 1995, incorporated in the present as a reference). HEp-2 cells (ATCC CCL 23) were maintained in MEM (Life Technologies, Gaithersburg, MD) with 10% fetal bovine serum, 50 μg ml gentamicin sulfate and 2 mM glutamine. The Vero cells from step 150 below were maintained in serum free medium VP-SFM (Formula No. 96-0353SA, Life Technologies) with 50 μg ml of gentamicin sulfate and 2 mM glutamine.

Isolation of RNA Virion, Reverse Transcription and PCR Amplification of Viral Genes, and Automated Sequencing To clone the viral genes or to verify the genetic markers of the recombinant chimeric viruses, the viruses were amplified on cultured cells and concentrated by precipitation with polyethylene glycol as described. previously described (Mbiguino et al., J. Virol. Methods 31: 161-170, 1991, incorporated herein by reference). The virion RNA was extracted from the virus granule using Trizol reagent (Life Technologies) and used as the template for reverse transcription (RT) with the Superscript Preamplification system (Life Technologies). The cDNA was amplified by PCR additionally using the Advantage cDNA equipment (Clontech, Palo Alto, CA).

For cloning or for sequencing purposes / the DNA amplified by RT-PCR was purified from agarose gels using a NA45 DEAE membrane as suggested by the manufacturer (Schleicher &Schuell, Keene, NH). Sequencing was performed with the rodaminad dye terminator cycle sequencing kit (Perkin Elmer, Forster City CA) and an ABI 310 Gene Analyzer (Perkin Elmer, Forster City, CA).

Construction of the chimeric PIV3-PIV2 antigenomic DNAs encoding the F and HN proteins of full PIV2 or the chimeric F and HN proteins containing an ectodomain derived from PIV2 and a cytoplasmic tail domain derived from PIV3 A DNA encoding a Full length PIV3 antigenomic RNA was constructed in which the ORF F and HN of PIV3 were replaced by their PIV2 counterparts following the strategy described above. { Tao et al., J. Virol. 72: 2955-2961, 1998) for PIV3-PIV1. The details of this construction are presented in Figure 17. The PIV2 / V94 propagated in Vero cells was concentrated and virion RNA (vRNA) was extracted from the virus granule using Trizol reagents. The FIV and FN ORFs of PIV2 / V94 were reverse transcribed from the vRNA using random hexamer primers and the SuperScript Preamplification system before being amplified by PCR using the Advantage cDNA kit and the primer pairs specific for the F and HN genes of PIV2, respectively (1, 2 and 3, 4, Table 22). The amplified cDNA fragment from ORF F of PIV2 was digested with Ncol plus Bamm and ligated into the Ncol-Bamm window of pLit.PIV31.Fhc. { Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference) to generate pLit. PIV32Fhc. the BspEI site in the full length cDNA of PIV3 is unique and was planned to be used to exchange segments between the cDNAs (see Figures 17 to 19). Therefore, a BspEI site that was found in the ORF F of PIV2 was removed by site-directed mutagenesis without affecting the amino acid sequence. The cDNA fragment of the PIV2 HN ORF was digested with Ncol plus HindIII and ligated into the NcoI-HindIII window of pLit.PIV31.HNhc (Tao et al., J. Virol. 72: 2955-2961, 1998) to generate pLit. .PIV32HNhc. The ORF of PIV2 in pLit. PIV32Fhc and pLit. PIV32HNhc were sequenced and the sequence was found to be as designated. The nucleotide sequences for the ORF F and HN of PIV2 were presented in GenBank. pLit. PIV32Fhc and pLitPIV32HNhc each were digested with PpuMI plus Spel and assembled to generate pLitPIV32hc. The BspEI-Spel 4 kb fragment from pLitPIV32hc was introduced into the BspEI-Spel window of p38'APIV31hc (Skiadopoulos et al., J. Virol. 18: 503-510, 1999, incorporated herein by reference) to generate p38'APIV32hc. The 6.5 kb fragment, generated by the BspEI and Sphl digestion of p38 'APIV32hc, containing the full length ORF and HN of PIV2 was introduced into the BspEI-Sphl window of pFLC.2G + .hc (Tao et al., J Virol 72: 2955-2961, 1998) to generate pFLC.PIV32hc (Figure 17, Table 23 = SEQ ID NO.60).

Table 22. Primers used in the construction of full-length chimeric antigenic cDNAs PIV3-2 Ceba- Gen Position direction Used in Sequence3rd construction or No. characterization of: Start Term 1 ptV7 F Direction Initial coil F 20 pb in the a ccATGaATCACCTGCATCCAAT of PIV2 5070 address 3 '5091 pFLC.PIV32hc (SEQ ID NO. 40) PIV2 F antisense Finishing coil 20 bp in the t ^ KMccTAAOATATCCCATATATQTTTC F of PIV2 6732b direction 5 '6705b pFLC.PIV32hc (SEQ ID NO.41) PIV2 H direction Initial cell F 18 bp in the _. 2-tgCCATCGAAGATTACAGCAAT of PIV2 6837b address 3 '6856b pFLC.PIV32hc (SEQ ID NO.19) PIV2 HN antisense Finalizing codon 17 bp in the _nataagcTTAAAGCATTAGTTCCC F of PIV2 8558b 5'8538b address pFLC.PIV32hc (SEQ ID NO.20) 5 PIV2 F direction 5069 »5088 * ATGCATCACCTGCATCCAAT pFLC.PI V32TM (SEQ jp NO, 42) 6 PIV2 F antisense 6538s is go TAGTGAATAAAGTGTCTTGGCT pFLC.Pl V32TM (SEQ ID NO.43) 7 PIV2 HN direction 6962 * 6985 *, CATGAGATAATTCATCTTGATGTT pFLC.PIV32TM (SEQ ID NO.44) 8 PIV2 H _ ". agcTTAAAGCATTAGTTCCCTTAA antisense 8560 ° 8537 * PFLC.PIV32TM (SEQ jD NO 4Jj _ _ ATCATAATTATTTTGATAATGATCATTA 9 PIV3 F direction 6539 * 6566 * PFLC.PIV32TM (SEQ ID NO.46) -, ..., "._. GTTCAGTGCTTGTTGTGTT 10 PIV3 F antisense 5068e 5050 * pFLC.PIV32TM jggq ro NQ 47J "" "TCATAATTAACCATAATATGCATCAAT I I PIV3 HN direction 856G 8587 * PFLCPIV32TM (SEQ ID NO.48) 12 PIV3 HN antisense 6938 * pFLC.PlV32TM GA GGAATTAATTAOCACTATGAT (SEQ ID NO.49) 13 PIV2 F direction 5069 * 5088 'pFLC.PIV32CT ATGCATCACCTGCATCCAAT (SEQ IDNO.50) 14 PIV2 F antisense $ 607 * «589 * GATGATGTAGGCAATCAGC pFLC.PIV32CT ( SEQ IDNO 51) 15 P1V2 HN direction «« 87 * 6904 * ACTGCCACAATTCTTGGC pFLC.PIV32CT (SEQ ID NO 52) 16 P1V2 H antisense g536J 8511 * TTAAAGCATTAGTTCCCTTAAAAATG pFLC.PlV32CT (SEQ ID NO.53) 17 PIV3 F sense ^? * 6642 * AAGTATTACAGAATTCAAAAGAG (SEQ ID NO.54) pFLC.PlV32CT? ig P1V3 F antisense ^ 5050 * GTTCAGTGCTTGTTGTGTT n (SEQ ID NO.47) pFLC.PIV32CT 19 PIV3 HN direction e523 «8551 * TCATAATTAACCATAATATGCATCAAT (SEQ ID NO.48) pFLC.PIV32CT 20 PIV3 H antisense ¿gg« 6879 * CTTATTAGTGAGCTTGTTGC (SEQ ID NO.55) 21 P1V2 F direction 6608 * pFLC.PIV32CT 6630 * · * ACCGCAGCTGTAGCAATAGT (SEQ ID NO.56) 22 PIV2 HN antisense 7502 * confirmation GATTCCATCACTTAGGTAAAT 7501 '7481 * of the chimera (SEQ ID NO.57) 23 PIV3 M sense 4? J ^ 4 4780 * GATACTATCCTAATATTATTGC confirmation of the chimera (SEQ ID O.58.} Antisense PIV3 L 9100 * 9081 * confi mation GCTAATTTTGATAGCACATT 9076 * 9057 * of the chimera (SEQ ID NO 59) All primers were noted as the PIV-specific sequences are uppercase / low-PIV sequences in lowercase, the initial and final codons in bold and the restriction sites underlined. The numbers are in the nt positions in the pFLC.PIV32hc construct of the full-length antigenomic cDNA. The numbers are in the nt positions in the pFLC. PIV32TM and pFLC. PIV32TMcp45 of the construction of full-length antigenomic cDNA. The numbers are in the nt positions in the pFLC. PIV32CT and pFLC. PIV32CTcp45 of the full-length antigenomic cDNA construct.

TABLE 23 (SEQ ID NO 60) Sequence of pFLC.PIV32, 15492 base pairs in sense orientation (only the insert is shown) 1 ACCAAACAAG AGAAGAAACT TGTCTGGGAA TATAAATTTA ACTTTAAATT AACTTAGGAT 61 TAAAGACATT GACTAGAAGG TCAAGAAAAG GGAACTCTATATATTTCAAAA ATGTTGAGCC 121 TATTTGATAC ATTT TGCA CGTAGGCAAG AAAACATAAC AAAATCAGCC GGTGGAGCTA 181 TCATTCCTG6 ACAGAAAAAT ACTGTCTCTA TATTCGCCCT TGGACCGACA ATAAC GATG 241 ATAATGAGAA AATGACATTA GCTCTTCTAT TTCTATCTCA TTCACTAGAT AATGAGAAAC 301 AACATGCACA AA6GGCAGGG TTCTTGGTGT CTTTATTGTC AATGGCTTAT GCCAATCCAG 361 AGCTCTACCT AACAACAAAT GGAAGTAATG CAGATGTCAA GTATGTCATA TACATGATTG 421 AGAAAGATCT AAAACGGCAA AAGTATGGAG GATTTGTGGT TAAGACGAGA GAGATGATAT 481 ATGAAAAGAC AACTGATTGG ATATTTGGAA GTGACCTGGA TTATGATCAG GAAACTATGT 541 TGCAGAACGG CAGGAACAAT TCAACAATTG AAGACCT GT CCACACATTT GGGTATCCAT 601 CATGTTTAGG AGCTC TATA ATACAGATCT GGATAGTTCT GGTCAAAGCT ATCACTAGTA 661 TCTCAGGGTT AAGAAAAGGC TTTTTCACCC GATTGGAAGC TTTCAGACAA GATGGAACAG 721 TGCAGGCAGG GCTGGTATTG AGCG GGACA CAGTGGATCA GATTGGGTCA ATCATGCGGT 761 CTCAACAGAG CTTGGTAACT CTTATGGTTG AAACAT AAT AACAATGAAT ACCAGCAGAA 841 ATGACCTCAC AACCATAGAA AAGAATATAC AAATTGTTGG CAACTACATA AGAGATGCAG 901 GTCTOGCTTC AITCTTCAAT ACAATCAGAT ATGGAATTGA GACCAGAAIG GCAGCTTTGA 961 CTCTATCCAC TCTCAGACCA GATATCAATA GATTAAAAGC TTTGATGGAA CTGTATTTAT 1021 CAAAGGGACC ACGCGCTCCT TTCATCTGTA TCCTCAGAGA TCCTATACAT GGTGAGTTCG 1081 CACCAGGCAA CTATCCTGCC ATATGGAGCT ATGCAATGGG GGTGGCAGTT GTACAAAATA 1141 GAGCCATGCA ACAGTATGTG ACGGGAAGAT CATATCTAGA CATTGATATG TTCCAGCAG 1201 GACAAGCAGT AGCACGTGAT GCCGAAGCTC AAATGAGCTC AACACTGGAA GATGAACTTG 1261 GAGTGACACA CGAATCTAAA GAAAGCTTGA AGAGACATAT AAGGAACATA AACAGTTCAG 1321 AGACATCTTT CCACAAACCG ACAGGTGGAT CAGCCATAGA GATGGCAATA GATGAAGAGC 1381 CAGAACAATT CGAACATAGA GCAGACAAG AACAAAATGG AGAACCCAA TCATCCATAA TTCAATATGC 1441 CTGGGCAGAA GGAAATAGAA GCGATGATCA GACTGAGCAA GCTACAGAAT 1501 CTGACAATAT CAAGACCGAA CAACAAAACA TCAGAGACAG ACAAACAAG AGACTCAACG 1561 ACAAGAAGAA ACAAAGCAGT CAACCACCCA CTAATOCCAC AAACAGAACA AACCAGGACG 1621 AAATAGATGA TCTGTTTAAC GCATTTGGAA GCAACTAATC GAATCAACAT TTTAATCTAA 1681 ATCAATAATA AATAAGAAAA ACTTAGGATT AAAGAATCCT ATCATACCGG AATATAGGGT 1741 GGAAATTTA GASTCTGCTT GAAACTCAAT CAATAGAGAG TTGATGGAAA GCGATGCTAA 1801 AAACTATCAA ATCAXGGATT CTTGGGAAGA GGAATCAAGA GATAAATCAA CTAATATCTC 1861 CTCGGCCCTC AACATCATTG AATTCATACT CAGCACCGAC CCCCAAGAAG ACTTATCGGA 1921 AAACGACACA ATCAACACAA GAACCCAGCA ACTCAGTGCC ACCATCTGTC AACCAGAAAT 1981 CAAAOCAACA GAAACAAGTG AGAAAGATAG TGGATCAACT GACAAAAATA GACAGTCCGG 2041 GTCATCACAC GAATGTACAA CAGAAGCAAA.AGATAGAAAT ATTGATCAGG AAACTGTACA 2101 GAGAGGACCT GGGAGAAGAA GCAGCTCAGA TAGTAGAGCT GAGACTGTGG TCTCTGGAGG 2161 AATCCCCAGA AGCATCACAG ATTCTAAAAA TGGAACCCAA AACACGGAGG ATATTGATCT 2221 CAATGAAATT AGAAAGATGG ATAAGGACTC TATTGAGGGG AAAATGCGAC AATCTCCAAA 2281 TGTTCCAAGC GAGATATCAG GAAGTGATGA CATATTTACA ACAGAACAAA GTAGAAACAG 2341 TGATCATGGA AGAAGCCTGG AATCTATCAG TACACCTGAT ACAAGATCAA TAAGTGTTGT 2401 TACTGCTGCA ACACCAG ATG ATGAAGAAGA AATACTAATG AAAAATAGTA GGACAAAGAA 2461 AAGTTCTTCA ACACATCAAG AAGATGACAA AAGAATTAAA AAAGGGGGAA AAGGGAAAGA 2521 CTGGTTTAAG AAATCAAAAG ATACCGACAA CCAGATACCA ACATCAGACT ACAGATCCAC 2581 ATCAAAAGGG CAGAAGAAAA TCTCAAAGAC AACAACCACC AACACCGACA CAAAGGGGCA 2641 AACAGAAATA CAGACAGAAT CATCAGAAAC ACAATCCTCA TCAIGGAATC TCATCATCGA 2701 CAACAACACC GACCGGAACG AACAGACAAG CACAACTCCT CCAACAACAA CTTCCAGATC 2761 AACTTATACA AAAGAATCGA TCCGAACAAA CTCTGAATCC AAACCCAAGA CACAAAAGAC 2821 AAATGGAAAG GAAAGGAAGS ATACAGAAGA GAGCAATCGA TTTACAGAGA GGGCAATTAC 2881 TCTATTGCAG AATCTTGGTG TAATTCAATC CACATCAAAA CTAGATTTAT ATCAACACAA February 41 ACGAGTTGTA TGTGTAGCAA ATGTACTAAA CAATGTAGAT ACTGCATCAA AGATAGATTT 3001 CCTGGCAGGA TTAGTCATAG GGGTTTCAAT GGACAACGAC ACAAAATTAA CACAGATACA 3061 AAATGAAATG CTAAACCTCA AAGCAGATCT AAAGAAAATG GACGAATCAC ATAGAAGATT 3121 GATAGAAAAT CAAAGAGAAC AACTGTCATT GATCACGTCA CTAATTTCAA ATCTCAAAAT 3181 TATGACTGAG AGAGGAGGAA AGAAAGACCA AAATGAATCC AATGAGAGAG TATCCATGAT 3241 CAAAACAAAA TTGAAAGAAG AA AAGATCAA GAAGACCAGG TTTGACCCAC TTATGGAGGC 3301 ACAAGGCATT GACAAGAATA TACCCGATCT ATATCGACAT GCAGGAGATA CACTAGAGAA 3361 CGATGTACAA GTTAAATCAG AGATATTAAG TTCATACAAT GAGTCAAATG CAACAAGACT 3421 AATACCCAAA AAAGTGAGCA GTACAATGAG ATCACTAGTT GCAGTCATCA ACAACAGCAA 3481 TCTC CACAA A6CACAAAAC AATCATACAT AAACGAACTC AAACGTTGCA AAAATGATGA 3S41 AGAAGTATCT GAATTAAT6G ACATGTTCAA T6AAGAT6TC AACAATTGCC AATGATCCAA 3601 CAAAGAAACG ACACCGAACA AACAGACAAG AAACAACAGT AGATCAAAAC CTGTCAACAC 3661 ACACAAAATC AAGCAGAATG AAACAACAGA TATCAATCAA TATACAAATA ACAAAAACTT 3721 AGGATTAAAG AATAAATTAA TCCTTGTCCA AAATGAGTAT AACTAACTCT GCAATATACA 3781 CATT C AGA ATCATCATTC TCTGAAAATG GTCATATAGA ACCATTACCA CTCAAAGTCA 3841 ATGAACAGAG GAAAGCAGTA CCCCACATTA GAGTTGCCAA GATCGGAAAT CCACCAAAAC 3901 ACGGATCCCG GTATTTAGAT GTCTTCTTAC TCGGCTTCTT CGAGATGGAA CGAATCAAAG 3961 ACAAATACGG GAGTGTGAAT GATCTCGACA GTGACCCGAG TTACAAAGTT TGTGGCTCTG 4021 GATCATTACC AATOGGATTG GCTAAGTACA CTGGGAATGA CCAGGAA TG TTACAAGCCG 4081 CAACCAAACT GGATATAGAA GTCAGAA GAA CAGTCAAAGC GAAAGAGATG GTTGTTTACA 4141 CGGTACAAAA TATAAAACCA GAACTGTACC CATGG CCAA TAGACTAAGA AAAGGAATGC 4201 TGTTCGATGC CAACAAAGTT GCTCTTGCTC CTCAATGTCT TCCACTAGAT AGGAGCATAA 4261 AATTTAGAGT AATCTTCGTG AATTGTACGG CAATTGGATC AATAACCTTG TTCAAAATTC 4321 CTAAGTCAAT GGCATCACTA TCTCTACCCA ACACAATATC AATCAATCTG CAGGTACACA 4381 TAAAAACAGG GGTTCAGACT GATTCTAAAG GGATAGTTCA AATTTTGGAT GAGAAAGGCG 4441 AAAAATCACT GAA TTCATG GTCCATCTCG GATTGATCAA AAGAAAAGTA GGCAGAATGT 501 ACTCTGTTGA ATACTGTAAA CAGAAAATCG AGAAAATGAG ATTGATATTT TCT TAGGAC 4561 TAGTTGGAGG AATCAGTCTT CATGTCAATG CAACTGGGTC CATATCAAAA ACACTAGCAA 4621 GTCAGCTGGT ATTCAAAAGA GAGATTTGTT ATCCTTTAAT GGATCTAAAT CCGCATCTCA 4681 ATCTAGTTAT CTGGGCTTCA TCAGTAGAGA TTACAAGAGT GGATGCAATT TTCCAACCTT 4741 CTT ACCTGG CGAGTTCAGA TACTATCCTA ATATTATTGC AAAAGGAGT GGGAAAATCA 801 AACAATGGAA CTAGTAATCT CTATTTTAGT CCGGACGTAT CTATTAAGCC GAAGCAAATA 4861 AAGGATAATC AAAAACTTAG GACAAAAGAG GTCAATACCA ACAACTATTA GCAGTCACAC 921 TCGCAAGAAT AAGAGAGAAG GGACCAAAAA AGTCAA ATAG GAGAAATCAA AACAAAAGGT 4981 ACACAACACC AGAACAACAA AATCAAAACA TCCAACTCAC TCAAAACAAA AATTCCAAAA 504 GAGACCGGCA ACACAACAAG CACTGAACAC CATGGATCAC CTGCAICCAA TGATAGTATG 5101 CATTTTTGTT ATGTACACTG GAATTGTAGG TTCAGATGCC ATTGCTGGAG ATCAAC CCT 5161 CAATGTAGGG GTCATTCAAT CAAAGATAAG ATCACTCATG TAC ACACTG ATGGTGGCGC 5221 TAGCTTTATT GTTGTAAAAT TACTACCCAA TCTTCCCCCA AGCAATGGAA CATGCAACAT 5281 CAOCAGTCTA GATGCATATA ATGTTACCCT ATTTAAGTTG CTAACACCCC TGATTGAGAA 5341 CCTGAGCAAA ATT C GCTG T ACAGATAC CAAACCCCGC CGAGAACGAT TTGCAGGAG 5401 CGTTATTGGG CTTGCTGCAC TAGGAGTAGC TACAGCTGCA CAAATAACCG CAGCTG AGC 5461 AATAGTAAAA GCCAATGCAA ATGCTGCTGC GATAAACAAT CTTGCATCTT CAATTCAATC 5521 CACCAACAAG GCAGTATCCG ATGTGATAAC TGCATCAAGA ACAATTGCAA CCGCAGTTCA 5581 AGCGATTCAG GATCACATCA ATGGAGCCAT TGTCAACGGG ATAACATCTG CATCATGCCG 5641 TGCCCATGA? 6CACTAATTG CGTCAATATT AAATTTGTAT CTCACTGACC TTACTACAAT 5701 ATTTCATAAT CAAATAACAA ACCCTGCGCT GACACCACTT TCCATCCAAG CTTTAAGAAT 5761 CCTCCTCGGT AGCACCTTGC CAATTGTCAT TGAATCCAAA CTCAACACAA AACTCAACAC 5821 AGCAGAGCTG CTCAGTAGCC GACTGTTAAC TGGTCAAATA ATTTCCATTT CCCCAATGTA 5881 CATGCAAATG CTAATTCAAA TCAATGTTCC GACATTTATA ATGCAACCCG GTGCGAAGGT 5941 AATTGATCTA ATTGCTATCT CTGCAAACCA TAAATTACAA GAAGTAGTTG TACAAGTTCC 6001 TAATAGAATT CTAGAATATG CAAATGAACT ACAAAACTAC CCAGCCAATG ATTGTTTCGT 6061 GACACCAAAC TCTGTATTTT GTAGATACAA TGAGGGTTCC CCGATCCCTG AATCACAATA 6121 TCAATGCTTA AGGGGGAATC TTAATTCTTG CACTTTTACC CCTATTATCG GGAACTTTCT 6181 CAAGCGATTC GCATTTGCCA ATGGTGTGCT CTATGCCAAC TGCAAATCTT TGCTATGTAA 6241 GTGTGCCGAC CCTCCCCATG. TTGTGTCTCA AGATGACAAC CAAGGCATCA GCATAATTGA 6301 TATTAAGAGG TGCTCTGAGA TGATGCTTGA CACTTTTTCA TTTAGGATCA CATCTACATT 6361 CAATGCTACA TACGTGACAG ACTTCTCAAT GATTAATGCA AATATTGTAC ATCTAAGTCC 6421 TCTAGACTTG TCAAATCAAA TCAATTCAAT AAACAAATCT CTTAAAAGTG CTGAGGATTG 6481 GATTGCAGAT AGCAACTTCT TCGCTAATCA AGCCAGAACA GCCAAGACAC TTTATTCACT 6541 AAGTGCAATC GCATTAATAC TATCAGTGAT TACTTTGGTT GTTGTGGGAT TGCTGATTGC 6601 CTACATCATC AAGCTGGTTT CTCAAATCCA TCAATTCAGA GCACTAGCTG CTACAACAAT 6661 GTTCCACAGG GAGAATCCTG CCGTCTTTTC CAAGAACAAT CATGGAAACA TATATGGGAT 6721 ATCTTAGGAT CCCTACAGAT CATTAGATAT TAAAATTATA AAAAACTTAG GAGTAAAGTT 6781 ACGCAATCCA ACTCTACTCA TATAATTGAG GAAGGACCCA ATAGACAAAT CCAAATCCAT 6841 GGAA5ATTAC AGCAATCTAT CTCTTAAATC AATTCCTAAA AGGACATGTA GAATCATTTT 6901 CC6AACTGCC ACAATTCTTG GCATATGCAC ATTAATTGTG CTATGTTCAA GTATTCTTCA 6961 TGAGATAATT CATCTTGATG TTTCCTCTGG TCTTATGAAT TCTGATGAGT CACAGCAAGG 7021 CATTATTCAG CCTATCATAG AATCATTAAA ATCATTGATT GCTTTGGCCA ACCAGATTCT 7081 ATATAATGTT GCAATAGTAA TTCCTC TTAA AATTGACAGT ATCGAAACTG TAATACTCTC 7141 TGCTTTAAAA GATATGCACA CCGGGAGTAT GTCCAATGCC AACTGCACGC CAGGAAATCT 7201 GCTTCTGCAT GATGCAGCAT ACATCAATGG AATAAACAAA TTCCTTGTAC TTGAATCATA 7261 CAATGGGACG CCTAAATAT6 6ACCTCTCCT AAATATACCC AGCTTTATCC CCTCAG AAC 7321 ATCTCCCCAT GGGTGTACTA GAATACCATC ATTTTCACTC ATCAAGACCC ATTGGTGTTA 7381 CACTCACAAT GTAATGCTTG GAGATTGTCT TGATTTCACG GCATCTAACC AGTATTTATC 7441 AATGGGGATA ATACAACAAT CTGCTGCAGG GTTTCCAATT TTCAGGACTA TGAAAACCAT 7501 TTACCTAAGT GATGGAATCA ATCGCAAAAG CTGTTCAGTC ACTGCTATAC CAGGAGGTTG 7561 TGTCTTGTAT TGCTATGTAG CTACAAGGTC TGAAAAAGAA GATTATGCCA CGA TGATCT 7621 AGC GAAC G AGAC TGCTT TCTATTATTA TAATGATACC TTTATTGAAA GAGTCATATC 7681 TCTTCCAAAT ACAACAGGGC AGTGGGCCAC AATCAACCCT GCA6TCGGAA GCGGGATCTA 7741 TCATCTAGGC TTTATCTTAT T CCTGTATA TGGTGGTCTC ATAAATGGGA CTACTTCTTA 7801 CAATGAGCAG TCCTCACGCT ATTTTATCCC AAAACATCCC AACATAACTT GTGCCGGAA 7861 CTCCAGCAAA CAGGCTGC TAGCACGGAG TTCCTATGTC ATCCGTTATC ACTCAAACAG 7921 GTAATTCAG AGTGCTGTTC TTATTTGTCC ATTG TCTGAC ATGCATAGAG AAGAGTGTAA 7981 TCTAGTTATG TTTAACAATT CCCAAGTCAT GATGGGTGCA GAAGGTAGGC TCTATGTTAT 8041 TGGTAATAAT TTGTATTATT ATCAACGCAG TTCCTCTTGG TGGTCTGCAT CGCTCTTTTA B101 CAGGATCAAT ACAGATTTTT CTAAAGGAAT TCCTCCGATC ATTGAGGCTC AATGGGTACC 8161 GTCCTATCAA GTTCCTCG C CTGGAGTCAT GCCATGCAAT GCAACAAGTT TT GCCCTGC 8221 TAATTGCATC ACAGGGGTGT ACGCAGATGT GTGGCCGCTT AATGATCCAG AACTCATGTC 8281 ACGTAATGCT CTGAACOOCA ACTATCGATT GCTGGAGCC TTTCTCAAAA ATGAGTCCAA 8341 CCGAACTAAT CCCACATTCT ACACTGCATC GGCTAACTCC CTCTTAAATA CTACCGGATT 8401 CAACAACACC AATCACAAAG CAGCATATAC ATC TCAACC TGCTTTAAAA ACACTGGAAC August 61 CCAAAAAATT TATTGTTTAA TAATAATTGA AATGGGCTCA TCTCTTTTAG GGGAGTTCCA 8521 AATAATACCA TTTTTAAGGG AA TAATGCT TTAAGCTTAA TTAACCATAA TATGCATCAA 8581 TCTATCTATA ATACAAGTAT ATGATAAGTA ATCTGCAATC AGACAATAGA CAAAAGGGAA 8841 ATATAAAAAA CTTAGGAGCA AAGCGTGCTC GGGAAATGGA CACTGAATCT AACAATGGCA 8701 CTGTATCTGA CATACTCTAT CCTGAGTGTC ACCTTAACTC TCCTATCGTT AAAGGTAAAA 8761 lAGCACAATT ACACACTATT ATGAGTCTAC CTCAGCCTTA TGATATGGAT GACGACTCAA 8821 TACTAGTTAT CACTAGACAG AAAATAAAAC TTAATAAATT GGATAAAAGA CAACGATCTA 8881 TTAGAAGATT AAAATTAATA TTAACTGAAA AAGTGAATGA CTTAGGAAAA TACACATTTA 89 1 TCAGATATCC AGAAATGTCA AAAGAAATGT TCAAATTATA TATACCTGGT ATTAACAGTA 9001 AAGTGACTGA ATTATTACTT AAAGCAGATA GAACATATAG TCAAATGAC GATGGATTAA 9061 GAGATCTATG GATTAATGTG CTATCAAAAT TAGCCTCAAA AAATGATGGA AGCAATTATG 9121 ATCTTAATGA AGAAATTAAT AATATATCGA AAGTTCACAC AACCTATAAA TCAGATAAAT 9181 GGTATAATCC ATTCAAA ACA TGGTTTACTA TCAAGTATGA TATGAGAAGA T ACAAAAAG 9241 CTCGAAATGA GATCACTTTT AATGTTGGGA AGGATTATAA CTTGTTAGAA GACCAGAAGA 9301 ATTTCTTATT GATACATCCA GAATTGGTTT TGATATTAGA TAAACAAAAC TATAATGGTT 9361 ATCTAATTAC TCCTGAATTA GTATTGATGT ATTGTGACGT AGTCGAAGGC CGATGGAATA 9421 TAAGTGCATG TGCTAAGTTA GATCCAAAAT TACAATCTAT GTATCAGAAA GGTAATAACC 9481 TGTGGGAAGT GATAGATAAA TTGTTTCCAA TTATGGGAGA AAAGACATTT GATGTGATAT 9341 CGTTATTAGA ACCACTTGCA TTATCCTTAA TTCAAACTCA TGATCCTGTT AAACAACTAA 9601 GAGGAGCTTT TTTAAATCAT GTGTTATCCG AGATGGAATT AATATTTGAA TCTAGAGAAT 9661 CGATTAAGGA ATTTCTGAGT GTAGATTACA TTGATAAAAT TTTAGATATA TTTAATAAGT 9721 CTACAATAGA TGAAATAGCA GAGATTTTCT CTTTTTTTAG AACATTTGGG CATCCTCCAT 9781 TAGAAGCTAG TATTGCAGCA GAAAAGGTTA GAAAATATAT GTATATTGGA AAACAATTAA 9841 AATTTGACAC TATTAATAAA TGTCATGCTA TCTTCTGTAC AATAATAATT AACGGATATA 9901 GAGAGAGGCA TGGTGGACAG TGGCCTCCTG TGACATTACC TGATCATGCA CACGAATTCA 9961 TCATAAATGC TTACGGTTCA AACTCTGCGA TATCATATGA AAATGCTGTT GATTATTACC 10021 AGAGCTTTAT AGGAATAAAA T TCAATAAAT TGATAGAGCC TCAGTTAGAT GAGGATTTGA 10081 CAATTTATAT GAAAGATAAA GCATTATCTC CAAAAAAATC AAATTGGGAC ACAGTTTATC 10141 CTGCATCTAA TTTACTGTAC OGTACTAACG CATCCAACGA ATCACGAAGA TTAGTTGAAG 10201 TATTTATAGC AGATAGTAAA TTTGATCCTC ATCAGATATT GGATTATGTA GAATCTGGGG 10261 ACTGGTTAGA TGATCCAGAA TTTAATATTT CTTATAGTCT TAAAGAAAAA GAGATCAAAC 10321 AGGAAGGTAG ACTCTTTGCA AAAATGACAT ACAAAATGAG AGCTACACAA GTTTTATCAG 10381 AGACCCTACT TGCAAATAAC ATAGGAAAAT TCTTTCAAGA AAATGGGATG GTGAAGGGAG 10441 AGATTGAATT ACTTAAGAGA TTAACAACCA TATCAATATC AGGAGTTCCA CGGTATAATG 10501 AAGTGTACAA TAATTCTAAA AGCCATACAG ATGACCTTAA AACCTACAAT AAAATAAGTA 10561 ATCTTAATTT GTCTTCTAAT CAGAAATCAA AGAAATTTGA ATTCAAGTCA ACGGATATCT 10621 ACAATGATGG ATACGAGACT GTGAGCTGTT TCCTAACAAC AGATCTCAAA AAATACTGTC 10681 TTAATTGGAG ATATGAATCA ACAGCTCTAT TTGGAGAAAC TTGCAACCAA ATATTTGGAT 10741 TAAATAAATT GTTTAATTGG TTACACCCTC GTCTTGAAGG AAGTACAATC TATGTAGGTG 10801 ATCCTTACTG TCCTCCATCA GATAAAGAAC ATATATCATT AGAGGATCAC CCTGATTCTG 10861 GTTTTTACGT TCAT AACCCA AGAGGGGGTA AGAAGGATT TGTCAAAAA TTATGGACAC 10921 TCATATCTAT AAGTSCAATA CATCTAGCAG CTGTTAGAAT AGGCGTGAGG GTGACTGCAA 10981 TGGTTCAAGG AGACAATCAA GCTATAGCTG TAACCACAAG AGTACCCAAC AATTATGACT 11041 ACAGAGTTAA GAAGSAGATA GTTTATAAAG ATGTAGTGAG ATTTTTTGAT TCATTAAGAG 11101 AA6TGATG6A TGATCTAGGT CATGAACTTA AATTAAATGA AACGATTATA AGTAGCAAGA 11161 TGTTCATATA TAGCAAAACA ATCTATTATG ATGGGAGAAT TCTTCCTCAA GCTCTAAAAG 11221 CATTATCTAG ATGTSTCTTC TGGTCA6AGA CAGTAATAGA CGAAACAAGA TCAGCATCTT 11281 CAAATTTGGC AACATCATTT GCAAAAGCAA TTGAGAATGG TTATTCACCT GTTCTAGGAT 11341 ATGCATGCTC AATTTTTAAG AATATTCAAC AACTATATAT TGCCCTTGGG ATGAATATCA 11401 ATCCAACTAT AACACAGAAT ATCAGAGATC AGTATTTTAG GAATCCAAAT TGGATGCAAT 11461 ATGCCTCTTT AATACCT6CT AGTGTTGGGG GATTCAATTA CATGGCCATG TCAAGATGTT 11521 TTGTAAGGAA TATTGGTGAT CCATCAGTTG CCGCATTGGC TGATATTAAA AGATTTATTA 11581 AGGCGAATCT ATTASACCGA AGTBTTCTTT ATAGGATTAT GAATCAAGAA CCAGGTGAGT 11641 CATCTTTTT GGACfGGGCT TCAGATCCAT ATTCATGCAA TTTACCACAA TCTCAAAATA 11701 TAACCACCATATAACA6CAA GGAATGTATT ACAAGATTCA CCAAATCCAT 11761 TATTATCTGG ATTATTCACA AATACAATGÁ TAGAAGAAGA TGAAGAATTA GCTGAGTTCC 11821 TGATGGACAG GAAGGTAATT CTCCCTAGAG TTGCACATGA TATTCTAGAT AATTCTCTCA 11881 CAGGAATTAG AAATGCCATA GCTQGAATGT TAGATACGAC AAAATCACTA ATTCGGGTTG 11941 GCATAAATAC AGGAGGACTG ACATATAGTT TGTTGAGGAA AATCAGTAAT TACGATCTAG_12001_TACAATATGA AACACTAAGT AGGACTTTGC GACTAATTGT AAGTGATAAA ATCAAGTATG 12061 AA6ATAT6TG TTCGGTAGAC CTTGCCATAG CATTGCGACA AAAGATGTGG ATTCATTTAT 12121 CAGGAGGAAG GATGATAAGT GGACTTGAAA CGCCTGACCC ATTAGAATTA CTATCTGGGG 12181 TAGTAATAAC AGGATCAGAA CATTGTAAAA TATGTTATTC TTCAGATGGC ACAAACCCAT 12241 ATACTTGGAT GTATTTACCC GGTAATATCA AAATAGGATC AGCAGAAACA GGTATATCGT 12301 CATTAAGAGT TOCTTATTTT 66ATCAGTCA CTGATGAAAG ATCTGAAGCA CAATTAGGAT 12361 ATATCAAGAA TCTTAGTAAA CCTGCAAAAG CCGCAATAAG AATAGCAATG ATATATACAT 12421 GGGCATTTGG TAATGATGAG ATATCTTGGA TGGAAGCCTC ACAGATAGCA CAAACACGTG 12481 CAAATTTTAC ACTAGATAGT CTCAAAATTT TAACACCGGT AGCTACATCA ACAAATTTAT CA 12541 CACAGATT AAAGGATACT 6CAACTCAGA TGAAATTCTC CAGTACATCA TTGATCAGAG 12601 TCAGCAGATT CATAACAATG TCCAATGATA ACATGTCTAT CAAAGAAGCT AATGAAACCA 12661 AAGATACTAA TCTTATTTAT CAACAAATAA TGTTAACAGG ATTAAGTGTT TTCGAATATT 12721 TATTTAGATT AAAAGAAACC ACAGGACACA ACCCTATAGT TATGCATCTG CACATAGAAG 12781 ATGAGTGTTG TATTAAAGAA AGTTTTAATG ATGAACATAT TAATCCAGAG TCTACATTAG_12841_AATTAATTCG ATATCCTGAA AGTAATGAAT TTATTTATGA TAAAGACCCA CTCAAAGATG 12901 TGGACTTATC AAAACTTATG GTTATTAAAfi ACCATTCTTA CACAATTGAT ATGAATTATT 12961 GGGATGATAC TGACATCATA CATGCAATTT CAATATGTAC TGCAATTACA ATAGCAGATA 13021 CTATGTCACA ATTAGATCGA GATAATTTAA AAGAGATAAT AGTTATTGCA AATGATGATG 13081 ATATTAATAG CTTAATCACT GAATTTTTGA CTCTTGACAT ACTTGTATTT CTCAAGACAT 13141 TTGGTGGATT ATTA6TAAAT CAATTTGCAT ACACTCTTTA TAGTCTAAAA ATAGAAGGTA 13201 GGGATCTCAT TTGGGATTAT ATAATGASAA CACTGAGAGA TACTTCCCAT TCAATATTAA 13261 AAGTATTATC TAATGCATTA TCTCATCCTA AAGTATTCAA GAGGTTCTGG GATTGTGGAG 13321 TTTTAAACCC TATTTATGGT CCTAATACTG CTAGTCAAGA CCAGATAAAA CTTGCCCTAT 13381 CTATATGTGA ATATTCACTA GATCTATTTA TGAGAGAÁTG GTTGAATGGT GTATCACTTG 13441 AAATATACAT TTGTGACAGC GATATGGAAG TTGCAAATGA TAGGAAACAA GCCTTTATTT 13501 CTAGACACCT TTCATTTGTT TGTTG TTAG CAGAAATTGC ATCTTTCGGA CCTAACCTGT 13561 TAAAGTTAAC ATACTTGGAG AGACTTGATC TATTGAAACA ATATCTTGAA TTAAATATTA 13621 AAGAAGACCC TACTCTTAAA TATGTACAAA TATCTGGATT ATTAATTAAA TCGTTCCCAT 13681 CAACTGTAAC ATACGTAAGA AAGACTGCAA TCAAATATCT AAGGATTCGC GGTATTAGTC 13741 CACCTGAGGT AATTGATGAT TGGGATCCGG TAGAAGATGA AAATATGCTG GATAACATTS 13801 TCAAAACTAT AAATGATAAC TGTAATAAAG ATAATAAAGG GAATAAAATT AACAATTTCT 13861 GGGGACTAGC ACTTAAGAAC TATCAAGTCC TTAAAATCAG ATCTATAACA AGTGATTCTG 13921 ATGATAATGA TAGACTAGAT GCTAATACAA GTGGTTTGAC ACTTCCTCAA GGAGGGAATT 13981 ATCTATCGCA TCAATTGAGA TTATTCGGAA TCAACAGCAC TAGTTGTCTG AAAGCTCTTG 140 1 AGTTATCACA AATTTTAATG AAGGAAGTCA ATAAAGACAA GGACAGGCTC TTCCTGGGAG 14101 AAGGAGCAGG AGCTATGCTA GCATGTTATG ATGCCACATT AGGACCTGCA GTTAATTATT 14161 ATAATTCAGG TTTGAATATA ACAGATGTAA TTGGTCAACG AGAATTGAAA A TATTTCCTT 14221 CAGAGGTATC ATTAGTAGGT AAAAAATTAG GAAATGTGAC ACAGATTCTT AACAGGGTAA 14281 AAGTACTGTT CAATGGGAAT CCTAATTCAA CATGGATAGG AAATATGGAA TGTGA6AGCT 14341 TAATATGGAG TGAATTAAAT GATAAGTCCA TTGGATTAGT ACATTCTGAT ATGGAAGGAG 14401 CTATCGGTAA ATCAGAAGAA ACTGTTCTAC ATGAACATTA TAGTGTTATA AGAATTACAT 14461 ACTTGATTGG GGATGATGAT GTTGTTTTAJG TTTCCAAAAT TATACCTACA ATCACTCCGA 14521 ATTGGTCTAG AATACTTTAT CTATATAAAT TATATTGGAA AGATGTAAGT ATAATATCAC 14581 TCMAACTTC TAATCCTGCA TCAACAGAAT TATATCTAAT TTCGAAAGAT GCATATTSTA 14641 CTATAATGGA ACCTAGTGAA ATTGTTTTAT CAAAACTTAA AAGATTGTCA CTCTTGGAAG 14701 AAAATAATCT ATTAAAATGG ATCATTTTAT CAAAQAAGAG GAATAATGAA TGGTTACATC 14761 ATGAAATCAA AGAAGGAGAA AGAGATTATG GAATCATGAG ACCATATCAT ATGGCACTAC 14821 AAATCTTTGG ATTTCAAATC AATTTAAATC ATCTGGCGAA AGAATTTTTA TCAACCCCAG 14881 ATCTGACTAA TATCAACAAT ATAATCCAAA GTTTTCAGCG AACAATAAAG GATGTTTTAT 14941 TTGAATGGAT TAATATAACT CATGATGATA AGAGACATAA ATTAGGCGGA AGATATAACA 15001 TATTCCCACT GAAAAATAAG GGAAAGTTAA GACTGCTATC GAGA AGACTA GTATTAAGTT 15061 GGATTTCATT ATCATTATCG ACTCGATTAC TTACAGGTCG CTTTCCTGAT GAAAAATTTG 1312 AACATAGAGC ACAGACTGGA TATGTATCAT TAGCTGATAC TGATTTAGAA TCATTAAAGT 15181 TATTGTCGAA AAACATCATT AAGAATTACA GAGAGTGTAT AGGATCAATA TCATATTGGT 1S241 TTCTAACCAA AGAAGTTAAA ATACTTATGA AATTGATCGG TGGTGCTAAA TTATTAGGAA 1S301 TTCCCAGACA ATATAAAGAA CCCGAAGACC AGTTATTAGA AAACTACAAT CAACATGATG 15361 AATTTGATAT CGATTAAAAC ATAAATACAA TGAAGATATA TCCTAACCTT TATCTTTAAG 15421 CCTAGGAATA GACAAAAAeT AAGAAAAACA TGTAATATAT ATATACCAAA CAGAGTTCTT 15481 CTCTTGTTTG GT strategy (Figure 18), the FF and HN ORFs of chimeric PIV3-PIV2, instead of the complete ORF exchange, were constructed in which the FIV and FN ORF regions of PIV2 coding for the ectodomains were amplified to from pLit. PIV32Fhc and pLit. PIV32HNhc, respectively, using PCR, Vent DNA polymerase (NEB, Beverly, A) and primer pairs specific for F (5, 6 in Table 22) and HN (7, 8 in Table 22) of PIV2. In parallel, the regions of the ORF F and HN of PIV3 coding for the ectodomains were deleted from their cDNA subclones pLit.PIV3.F3a and pLit.PIV3.HN4 (Tao et al., J. Virol. 72: 2955- 2961, 1998, incorporated herein by reference), respectively, using PCR, Vent DNA polymerase and primer pairs specific for F (9, 10 in Table 22) and HN (11, 12 in Table 22) of PIV3. The amplified F and HN cDNA fragments from PIV2 and PIV3 were purified from agarose gels and ligated to generate pLit. PIV32FTM and pLit. PIV32HNTM, respectively. The chimeric F and HN constructs were digested with PpuMI plus Spel and co-assembled together to generate pLit. PIV32TM, which was subsequently sequenced with the dRhodamine dye terminator sequencing kit through its specific PIV region in its entirety and was found to be as designed. The 4 kb fragment of BspEI Spel from pLit. PIV32TM was then introduced in the BspEI-Spel window of p38'APIV31hc to generate p38 'APIV32TM. The 6.5 kb BspEI-Sphl fragment of p38 'APIV32 ™, containing the chimeric F and HN genes of PIV3-PIV2 was introduced into the BspEI-Sphl window of pFLC.2G + .hc and pFLCcp45 (Skiadopoulos et al., J. Virol. 73: 1374-81, 1999, incorporated herein by reference) to generate pFLC. PIV32TM (Table 24; SEQ ID NO.61) and pFLC.PIV32TMcp45, respectively. The nucleotide sequence of the BspEI-Spel fragment, which contains the F and HN genes of chimeric PIV3-PIV2, was presented in GenBank.

TABLE 24 (SEQ ID No. 61) Sequence of pFLC. PIV32TM, 15498 base pairs in sense orientation (only the antigenome is shown) 1 ACCAAACAAG AGAAGAAACT TGTCTGGGAA TATAAATTTA ACTTTAAATT AACT AGGAT 61 TAAAGACATT CACTAGAAGG TCAAGAAAAG GCAACTCTAT AATTTCAAAA ATGTTGAGCC 121 TATTTGATAC ATTTAATGCA C6TA6GCAAG AAAACATAAC AAAATCAGCC GGTGGAGCTA 181 TCATTCCTGG ACAGAAAAAT ACTGTCTCTA TATTCGCCCT TGGACCGACA ATAACTGATG 24 ATAATGAGAA AATGACAT A GCTC TCTAT T CTATCTCA TTCACTAGAT AATGAGAAAC 301 AACATGCACA AAGGGCAGGG TTCTTGGTGT CTTTATTGTC AATGGCTTAT GCCAATCCAG 361 AGCTCTACCT AAGAACAAAT GGAAGTAATG CAGATG7CAA GTATGTCATA TACATGATTG 421 AGAAAGATCT AAAACGGCAA AAGTATGGAG GATTTG GG TAAGACGAGA GAGATGATAT 481 ATGAAAAGAC AACTGATTGG ATATTTGGAA GTGACCTGGA TTATGATCAG GAAACTATGT May 1 TGCAGAACGG CAGGAACAAT TCAACAATTG AAGACC7TGT CCACACATTT GGGTATCCAT 601 CATGTTTAGG AGCTCTTATA ATACAGATCT GGATAGTTCT GGTCAAAGCT ATCACTAGTA 661 TCTCAGGGTT AAGAAAAGGC TT TTCACCC GATTGGAAGC TTTCAGACAA GATGGAACAG 721 TGCAGGCAGG GCTGGTATTG AGCGGTGACA CAGTGGATCA GATTGGGTCA ATCATGCGGT 781 CTCAACAGAG CTTGGTAACT CTTATGGTTG AAACATTAAT AACAATGAAT ACCAGCAGAA 841 ATGACCTCAC AACCATAGAA AAGAATATAC AAATTGTTGG CAACT Acata AGAGATGCAG 901 GTCTCGCTTC ATTCTTCAAT ACAATCAGAT ATGGAATTGA GACCAGAATG GCAGCTT GA 961 CTCTATCCAC TCTCAGACCA GATATCAATA GATTAAAAGC TTTGATGGAA CTGTATTTAT 1021 CAAAGGGACC ACGCGCTCCT TTCATCTGTA TCCTCAGAGA TCCTATACAT GGTGAGTTCG 1081 CACCAGGCAA CTATCCTGOC ATATGGAGCT ATGCAATGGG GGTGGCAGTT GTACAAAATA 1141 GAGCCATGCA ACACTATGTG ACGGGAAGAT CATATC AGA CATTGATATG TTCCAGCTAG_1201_GACAAGCAGT AGCACGTGAT GCCGAAGCTC AAATGAGCTC AACACTGGAA GATGAACTTG 1261 GAGTGACACA CGAATCTAAA GAAAGCTTGA AGAGACATAT AAGGAACATA AACAGTTCAG 1321 AGA ATCrrC CCACAAACCG ACAGGTGGAT CAGCCATAGA GATGGCAATA GATGAAGAGC 1381 CAGAACAAT? CGAACATAGA GCAGATCAAG AACAAAATGG AGAACCTCAA TCATCCATAA 1441 TTCAATATGC CTGGGCAGAA GGAAA1AGAA GCGATGATCA GACTGAGCAA GCTACAGAAT 1501 CTGACAATAT CAAGACCGAA CAACAAAACA TCAGAGACAG ACTAAACAAG AGACTCAA G 1561 ACAAGAAGAA ACAAAGCAGT CAAOCACCCA CTAATCOCAC AAACAGAACA AACCAGGACG 1621 AAATAGATGA TCTGTTTAAC GCATTTGGAA GCAACTAATC GAATCAACAT TTTAATCTAA 1681 ATCAATAATA AATAAGAAAA ACTTAGGATT AAAGAATCCT ATCATACCGG AATATAGGGT 1741 GGTAAATTTA GAGTCTGCTT GAAACTCAAT CAATAGAGAG TTGATGGAAA GCGATGCTAA 1801 AAACTATCAA ATCATGGATT CTTGGGAAGA GGAATCAAGA GATAAATCAA C AATATC C 1861 CTCGGCCCTC AACATCATTG AATTCATACT CAGCACCGAC CCCCAAGAAG ACTTATCGGA 1921 AAACGACACA ATCAACACAA GAACCCAGCA ACtCAGTGCC ACCATCTGTC AACCAGAAAT 1981 CAAACCAACA GAAACAAGTG AGAAAGATAG TGGATCAACT GACAAAAATA GACAGTCCGG 2041 GTCATCACAC GAATGTACAA CAGAAGCAAA AGATAGAAAT ATTGATCAGG AAACTGTACA 2101 GAGAGGACCT GGGAGAAGAA GCAGCTCAGA TAGTAGAGCT GAGACTGTGG TCTCTGGAGG 2161 AATCCCCAGA AGCATCACAG ATTCTAAAAA TGGAACCCAA AACACGGAGG ATATTGATCT 2221 CAATGAAA.TT AGAAAGATGG ATAAGGACTC TATTGAGGGG AAAATGCGAC AATCTGCAAA 2281 TGTTCCAAfiC GAGATATCAG GAAGTGATGA CATATTTACA ACAGAACAAA GTAGAAACAG 2341 TGATCATGGA AGAAGCCTGG AATCTATCAG TACACCTGAT ACAAGATCAA TAAGTGTTGT 2401 TACTGCTGCA ACACCAGATG ATGAAGAAGA AATACTAATG AAAA ATAGTA GGACAAAGAA 2461 AAGTTCTTCA ACACATCAAG AAGATGACAA AAGAATTAAA AAAGGGGGAA AAGGGAAAGA 2321 CTGGTTTAAG AAATCAAAAG ATACCGACAA CCAGATACCA ACATCAGACT ACAGATCCAC 2581 ATCAAAAGGG CAGAAGAAAA TCTCAAAGAC AACAACCACC AACACCGACA CAAAGGGGCA 2641 AACAGAAATA CAGACAGAAT CATCAGAAAC ACAATCCTCA TCATGGAATC TCATCATCGA 2701 CAACAACACC GACCGGAACG AACAGACAAG CACAACTCCT CCAACAACAA CTTCCAGATC 2761 AACTTATACA AAAGAATCGA TCCGAACAAA CTCTGAATCC AAACCCAAGA CACAAAAGAC 2821 AAATGGAAAG GAAAGGAAGG ATACAGAAGA GAGCAATCGA TTTACAGAGA GGGCAATTAC 2881 TCTATTGCAG AATCTTGGTG TAATTCAATC CACATCAAAA CTAGATTTAT ATCAAGACAA 2941 ACGAGTTGTA TGTGTAGCAA ATGTACTAAA CAATGTAGAT ACTGCATCAA AGATAGATTT 3001 CCTGGCAGGA TTAGTCATAG GGGTTTCAAT GGACAACGAC ACAAAATTAA CACAGATACA 3061 AAATGAAATG CTAAACCTCA AAGCAGATCT AAAGAAAATG GACGAATCAC ATAGAAGATT 3121 GATAGAAAAT CAAAGAGAAC AACTGTCATT GATCACGTCA CTAATTTCAA ATCTCAAAAT 3181 TATGACTGAG AGAGGAGGAA AGAAAGACCA AAATGAATCC AATGAGAGAG TATCCATGAT 3241 CAAAACAAAA TTGAAAGAAG AAAAGATCAA GAAGACCAGG TTTGACCCAC TTATGGAGGG 3301 ACAAGGCATT GACAAGAATA TACCCGATCT ATATCGACAT GCAGGAGATA CACTAGAGAA 3361 CGATGTACAA GTTAAATCAG AGATATTAAG TTCATACAAT GAGTCAAATG CAACAAGACT 3421 AATACCCAAA AAAGTGAGCA GTACAATGAG ATCACTAGTT GCAGTCATCA ACAACAGCAA 3481 TCTCTCACAA AGCACAAAAC AATCATACAT AAACGAACTC AAACGTTGCA AAAATGATGA 3541 AGAAGTATCT GAATTAATGG ACATGTTCAA TGAAGATGTC AACAATTGCC AATGATCCAA 3601 CAAAGAAACG ACACCGAACA AACAGACAAG AAACAACAGT AGATCAAAAC CTGTCAACAC 3661 ACACAAAATC AAGCAGAATG AAACAACAGA TATCAATCAA TATACAAATA AGAAAAACTT 3721 AGGATTAAAG AATAAATTAA TCCTTGTCCA AAATGAGTAT AACTAACTCT GCAATATACA 3781 CATTCCCAGA ATCATCATTC TCTGAAAATG GTCATATAGA ACCATTACCA CTCAAAGTCA 3841 ATGAACAGAG GAAAGCAGTA CCCCACATTA GAGTTGCCAA GATCGGAAAT CCACCAAAAC 3901 ACGGATCCCG GTATTTAGAT GTCTTCTTAC TCGGCTTCTT CGAGATGGAA CGAATCAAAG 3961 ACAAATACGG GAGTGTGAAT GATCTCGACA GTGACCCGAG TTACAAAGTT TGTGGCTCTG 4021 GATCATTACC AATCGGATTG GCTAAGTACA CTGGGAATGA CCAGGAATTG TTACAAGCCG 4081 CAACCAAACT GGATATAGAA GTGAGAAGAA CAGTCAAAGC GAAAGAGATG GTTGT TTACA 4141 CGG ACAAAA TATAAAACCA GAACTGTACC CATGGTCCAA TAGACTAAGA AAAGGAATGC 4201 TGTTCGATGC CAACAAAGTT GCTCTTGCTC CTCAATGTCT TCCACTAGAT AGGAGCATAA 4261 AATTTAGAGT AATCTTCGTG AATTGTACGG CAATTGGATC AATAACCTTG TTCAAAATTC 4321 CTAAGTCAAT GGCATCACTA TCTCTACCCA ACACAATATC AATCAATCTG CAGGTACACA 4381 TAAAAACAGG GGTTCAGACT GATTCTAAAG GGATAGTTCA AATTTTGGAT GAGAAAGGCG April 41 AAAAATCACT 6AATTTCATG GTCCATCTCG GATTGATCAA AAGAAAAGTA GGCAGAATGT 4S01 ACTCTGTTGA ATACTGTAAA CAGAAAATCG AGAAAATGAG ATTGATATTT TCTTTAGGAC 4561 TAGTTGGAGG AATCAGTCTT CATGTCAATG CAACTGGGTC CATATCAAAA ACACTAGCAA 4621 GTCAGCTGGT ATTCAAAAGA GAGATTTGTT ATCCTTTAAT GGATCTAAAT CCGCATCTCA 4681 ATCTAGTTAT CTGGGCTTCA TCAGtAGAGA TTACAAGAGT GGATGCAATT TTCCAACCTT 4741 CTTTACCTGG CGAGTTCAGA TACTATCCTA ATATTATTGC AAAAGGAGTT GGGAAAATCA 4801 AACAATGGAA CTAGTAATCT CTATTTTAGT CCGGACGTAT CTATTAAGCC GAAGCAAATA 4861 AAGGATAATC AAAAACTTAG GACAAAAGAG GTCAATACCA ACAACTATTA GCAGTCACAC 4921 TOGCAAGAAT AAGAGAGAAG GGACCAAAAA AGTCAAATAG GAGAAATCAA AACAAAAGGT 4981AGAACAACAA AATCAAAACA TCCAACTCAC TCAAAACAAA AATTCCAAAA 5041 GAGACCGGCA ACACAACAAG CACTGAACAT GCATCACCTG CATCCAATGA TAGTATGCAT 5101 TTTTGTTATG TACACTGGAA TTGTAGGTTC AGATGCCATT GCTGGAGATC AACTCCTCAA 5161 TGTAGGGGTC ATTCAA CAA AGATAAGATC ACTCATGTAC TACACTGATG GTGGCGCTAG_5221_CTTTATTGTT GTAAAATTAC TACCCAATCT TCCCCCAAGC AATGGAACAT GCAACATCAC 5281 CAGTCTAGAT GCATATAATG TTACCCTATT TAAGTTGCTA ACACCCCTGA TTGAGAACCT S341 GAGCAAAATT TCTGCTGTTA CAGATACCAA ACCCCGCCGA GAACGATTTG CAGGAGTCGT 5401 TATTGGGCTT GCTGCACTAG GAGTAGCTAC AGCTGCACAA ATAACCGCAG CTGTAGCAAT 5461 AGTAAAAGCC AATGCAAATG CTGCTGCGAT AAACAATCTT GCATCTTCAA TTCAATCCAC 5521 CAACAAGGCA GTATCCGATG TGATAACTGC ATCAAGÁACA ATTGCAACCG CAGTTCAAGC 5581 GAT CAGGAT CACATCA TG GAGCCATTGT CAACGGGATA ACATCTGCAT CATGCCGTGC 5641 CCATGATGCA CTAATTGGGT CAATATTAAA TTTGTATCTC ACTGAGCTTA CTACAATATT 5701 TCATAATCAA ATAACAAACC C GCGCTGAC ACCACTTTCC ATCCAAGCTT TAAGAATCCT 5761 CCTCGGTAGC ACCTTGCCAA T GTCATTGA ATCCAAACTC AACACAAAAC TCAACACAGC 5821 AGAGC TGCTC AGTAGCGGAC TGTTAACTGG TCAAATAATT TCCATTTCCC CAATGTACAT 5881 GCAAATGC A ATTCAAATCA ATGTTCCGAC ATTTATAATG CAACCCGGTG CGAAGGTAAT 5941 TGATCTAATT GCTATCTCTG CAAACCATAA ATTACAAGAA GTAGTTGTAC AAGTTCCTAA 6001 TAGAATTCTA GAATATGCAA ATGAACTACA AAACTACCCA GCCAATGATT GTTTCGTGAC 6061 ACCAAACTCT GTATTTTGTA GATACAATGA GGGTTCCCCG ATCCCTGAAT CACAATATCA 6121 ATGCríAAGG GGGAATCTTA ATTCTTGCAC TTTTACCCCT ATTATCGGGA ACTTTCTCAA 6181 GCGATTCGCA TTTGCCAATG GTGTGCTC A TGCCAACTGC AAATCTTTGC TATGTAAGTG 6241 TGCCGACCCT CCCCATGTTG TGTCTCAAGA TGACAACCAA GGCATCAGCA TAATTGATAT 6301 TAAGAGGTGC TCTGAGATGA TGCTTGACAC TTTTTCATTT AGGATCACAT CTACATTCAA 6361 TGCTACATAC GTGACAGACT TCTCAATGAT TAATGCAAAT ATTGTACATC TAAGTCCTCT 6421 AGACTTGTCA AATCAAATCA ATTCAATAAA CAAATCTCTT AAAAGTGCTG AGGATTGGAT 6481 TGCAGATAGC AACTTCTTCG CTAATCAAGC CAGAACAGCC AAGACACTTT ATTCACTAAT 6541 CATAATTATT TTGATAATGA TCATTATATT GTTTATAATT AATATAACGA TAATTACAAT 6601 TGCAATTAAG TATTACAGAA TTCAAAAGAG AAATCGAGTG GATCAAAATG ACAAGCCATA 6661 TGTACTAACA AACAAATAAC ATATCTACAG ATCATTAGAT ATTAAAATTA TAAAAAACTT 6721 AGGAGTAAAG TTACGCAATC CAACTCTACT CATATAATTG AGGAAGGACC CAATAGACAA 6781 ATCCAAATTC GAGATGGAAT ACTGGAAGCA TACCAATCAC GGAAAGGATG CTGGTAATGA 6841 GCTGGAGACG TCTATGGCTA CTCATGGCAA CAAGCTCACT AATAAGATAA TATACATATT 6901 ATGGACAATA ATCCTGGTGT TATTATCAAT AGTCTTCATC ATAGTGCTAA TTAATTCCAT 6961 CCATGAGATA ATTCATCTTG ATGTTTCCTC TGGTCTTATG AATTCTGATG AGTCACAGCA 7021 AGGCATTATT CAGCCTATCA TAGAATCATT AAAATCATTG ATTGCTTTGG CCAACCAGAT 7081 TCTATATAAT GTTGCAATAG TAATTCCTCT TAAAATTGAC AGTATCGAAA CTGTAATACT 7141 CTCTGCTTTA AAAGATATGC ACACCGGGAG TATGTCCAAT GCCAACTGCA CGCCAGGAAA 7201 TCTGCTTCTG CATGATGCAG CATACATCAA TGGAATAAAC AAATTCCTTG TACTTGAATC 7261 ATACAATGGG ACGCCTAAAT ATGGACCTCT CCTAAATATA CCCAGCTTTA TCCCCTCAGC 7321 AACATCTCCC CATGGGTGTA CTAGAATACC ATCATTTTCA CTCATCAAGA CCCATTGGTG 7381 TTACACTCAC AATGTAATGC TTGGAGATTG TCTTGATTTC ACGGCATCTA ACCAGTATTT 7441 ATCAATGGGG ATAATACAAC AATCTGCTGC AGGGTTTCCA ATTTTCAGGA CTATGAAAAC 7501 CAT TACCTA AGTG ATGGAA TCAATCGCAA AAGCTGTTCA GTCACTGCTA TACCAGGAGG 7561 TTGTGTCTTG TATTGCTATG TAGCTACAAG GTCTGAAAAA GAAGATTATG CCACGACTGA 7621 TCTAGCTGAA CTGAGACTTG CTTTCTATTA TTATAATGAT ACCTTTATTG AAAGAGTCAT 7681 ATCTCTTCCA AATACAACAG GGCAGTGGGC CACAATCAAC CCTG AGTCG GAAGCGGGAT 7741 CTATCATCTA GGCTTTATCT TATT CCTGT ATATGGTGGT CTCATAAATG GGACTACTTC 7801 TTACAATGAG CAGTCCTCAC GCTATTTTAT CCCAAAACAT CCCAACATAA CTTGTGCCGG 7861 TAACTCCAGC AAACAGGCTG CAATAGCACG GAGTTCCTAT GTCATCCGTT ATCACTCAAA 7921 CAGGTTAATT CAGAGTGCTG TTCTTATTTG TCCATTGTCT GACATGCATA CAGAAGAGTG 7981 TAATCTAGTT ATGTTTAACA ATTCCCAAGT CATGATGGGT GCAGAAGGTA GGCTCTATGT 8041 TATTGGTAAT AATTTG ATT ATTAT AACG CAGTTCCTCT TGGTGGTCTG CATCGCTCTT "101 TTACAGGATC AATACAGATT TTTCTAAAGG AATTCCTCCG ATCATTGAGG CTCAATGGGT 8161 ACCGTCCTAT CAAGT CCTC GTCCTGGAGT CATGCCATGC AATGCAACAA GTTTTTGCCC 8221 TGCtAATTGC ATCACAGGGG TGTACGCAGA TGTGTGGCCG CTTAATGATC CAGAACTCAT 8281 GTCACGTAAT GCTCTGAACC CCAACTATCG ATTTGCTGGA GCCTTTCTCA AAAATGAGTC 8341 CAACCGAACT AATCCCACA TCTACACTGCTCCCTCTTAA ATACTACCGG 8401 ATTCAACAAC ACCAATCACA AAGCAGCATA TACATCTTCA AOCTGCTTTA AAAACACTGG August 61 AACCCAAAAA ATTTATTGTT TAATAATAAT TGAAATGGGC TCATCTCTTT TAGGGGAGTT 8521 CCAAATAATA CCATTTTTAA GGGAACTAAT GCTTTAAGCT TCATAATTAA CCATAATATG 8581 CATCAATCTA TCTATAATAC AAGTATATGA TAAGTAATCA GCAATCAGAC AATAGACAAA 8641 AGGGAAATAT AAAAAACtTA GGAGCAAAGC GTGCTCGGGA AATGGACACT GAATCTAACA 8701 ATGGCACTGT ATCTGACATA CTCTATCCTG AGTGTCACCT TAACTCTCCT ATCG AAAG 8761 GTAAAATAGC ACAA7TACAC ACTATTATGA GTCTACC CA GCCTTATGAT ATGGATGACG 8821 ACTCAATACT AGTTATCACT AGACAGAAAA TAAAACTTAA TAAATTGGAT AAAAGACAAC 8881 GATCTATTAG AAGATTAAAA TTAATATTAA CTGAAAAAGT GAATGACTTA GGAAAATACA 8941 CATTTATCAG ATATCCAGAA ATGTCAAAAG AAA GTTCAA ATTATATATA CCTGGTATTA 9001 ACAGTAAAGT GACTGAATTA TTACTTAAAG CAGATAGAAC ATATAGTCAA ATGACTGATG 9061 GATTAAGAGA TCTATGGATT AATGTGCTAT CAAAATTAGC CTCAAAAAAT GATGGAAGCA 9121 ATTATGATCT TAATGAAGAA ATTAATAATA TATCGAAAGT TCACACAACC TATAAATCAG 9181 ATAAATGGTA TAATCCATTC AAAACATGGT TTACTAT CAA GTATGATATG AGAAGATTAC 9241 AAAAAGCTCG AAATGAGATC ACTTTTAATG TTGGGAAGGA TTATAACTTG TTAGAAGACC 9301 AGAAGAATTT CTTATTGATA CATCCAGAAT TGGTTTTGAT AT AGATAAA CAAAACTATA 9361 ATGGTTATCT AATTACTCCT GAATTAGTAT TGATGTA TG TGAGGTAGTC GAAGGCCGAT 9421 GGAATATAAG TGCAT6TGCT AAGT AGATC CAAAATTACA ATCTATGTAT CAGAAAGGTA 9481 ATAACCTGTG GGAAGTGATA GATAAATTGT TTCCAATTAT GGGAGAAAAG ACATTTGATG 9541 TGATATCGTT ATTAGAACCA CTTGCATTAT CCTTAATTCA AACTCATGAT CCTGTTAAAC 9601 AACTAAGAGG AGCTTTTTTA AAT ATGTGT TATCCGAGAT GGAATTAATA TTTGAATCTA 9661 GAGAATCGAT TAAGGAATTT CTGAGTGTAG ATTACATTGA TAAAATTTTA GATATATTTA 9721 ATAAGTCTAC AATAGATGAA ATAGCAGAGA TTTTCTCTTT TTTTAGAACA TTTGGGCATC 9781 CTCCATTAGA AGCTAGTATT GCAGCAGAAA AGGT AGAAA ATATATGTAT ATTGGAAAAC 9841 AATTAAAATT TGACACTATT AATAAATGTC ATGCTATCTT CTGTACAATA ATAATTAACG 9901 GATATAGAGA GAGGCATGG GGACAGTGGC CTCCTGTGAC ATTACC GAT CATGCACACG 9961 AATTCATCAT AAATGCTTAC GGTTCAAACT CTGCGATATC ATATGAAAAT GCTGTTGATT 10021 ATTACCAGAG CTTTATAGGA ATAAAATTCA ATAAATTCAT AG AGCCTCAG TTAGATGAGG 10081 ATTTGACAAT TTATATGAAA GATAAAGCAT TATCTCCAAA AAAATCAAAT TGGGACACAG 10141 TTTATCCTGC ATCTAATTTA CTGTACCGTA CTAACGCATC CAACGAATCA CGAAGAT AG 10201 TTGAAGTATT TATAGCAGAT AGTAAATTTG ATCCTCATCA GATATTGGAT TATGTAGAAT 10261 CTGGGGACTG GTTAGATGAT CCAGAATTTA ATATTTCTTA TAGTCTTAAA GAAAAAGAGA 10321 TCAAACAGGA AGGTAGACTC TTTGCAAAAA TGACATACAA AA GAGAGCT ACACAAGTTT 10381 TATCAGAGAC CCTACTTGCA AATAACATAG GAAAATTCTT TCAAGAAAAT GGGATGGTGA 10441 AGGGAGAGAT TGAATTACTT AAGAGATTAA CAACCATATC AATATCAGGA GTTCCACGGT 10501 ATAATGAAGT GTACAATAAT TCTAAAAGCC ATACAGATGA CCTTAAAACC TAiCAATAAAA 10561 TAAGTAATCT TAATTTGTCT TCTAATCAGA AATCAAAGAA ATTTGAATTC AAGTCAACGG 10621 ATATCTACAA TGATGGATAC GAGACTGTGA GCTGTTTCCT AACAACAGAT CTCAAAAAAT 10681 ACTGTCTTAA TTGGAGATAT GAATCAACAG CTCTATTTGG AGAAACTTGC AACCAAATAT 10741 TTGGATTAAA TAAATTGTTT AATTGGTTAC ACCCTCGTCT TGAAGGAAGT ACAATCTATG 10801 TAGGTGATCC TTACTGTCCT CCATCAGATA AAGAACATAT ATCATTAGAG GATCACCCTG 10861 ATTCTGGTT TTACGTTCAT AACCCAAGAG GGGGT Ataga AGGATTTTGT CAAAAATTAT 10921 GGACACTCAT ATCTATAAGT GCAATACATC TAGCAGCTGT TAGAATAGGC GTGAGGGTGA 10981 CTGCAATGGT TCAAGGAGAC AATCAAGCTA TAGCTGTAAC CACAAGAGTA CCCAACAATT 11041 ATGACTACAC ASTTAAGAAG GAGATAGTTT ATAAAGATGT AGTGAGATTT TTTGATTCAT 11101 TAAGAGAAGT GATGGATGAT CTAGGTCATG AACTTAAATT AAATGAAACG ATTATAAGTA 11161 GCAAGATGTT CATATATAGC AAAAGAATCT ATTATGATGG GAGAATTCTT CCTCAAGCTC 11221 TAAAAGCATT ATCTAGATGT GTCTTCTGGT CAGAGACAGT AATAGACGAA ACAAGATCAG 11281 CATCTTCAAA TTTGGCAACA TCATTTGCAA AAGCAATTGA GAATGGTTAT TCACCTGTTC 11341 TAG_6_ATATGC ATGCTCAATT TTTAAGAATA TTCAACAACT ATATATTGCC CTTGGGATGA 11401 ATATCAATCC AACTATAACA CAGAATATCA GAGATCAGTA TTTTAGGAAT CCAAATTGGA 11461 TGCAATATGC CTCTTTAATA CCT6CTAGTG TTGGGGGATT CAATTACATG GCCATGTCAA 11521 GATGTTTTGT AAGGAATATT GGTGATCCAT CAGTTGCCGC ATTGGCTGAT ATTAAAAGAT 11581 TTATTAAGGC GAATCTATTA GAOOSAAGTG TTCTTTATAG GATTATGAAT CAAGAACCAG 11641 GTGAGTCATC TTTTTTGGAC TGGGCTTCAG ATCCATATTC ATGCAATTTA CCACAATCTC 11701 AAAATATAAC CACCATGATA AAAAATAT AATGTATTACAA GATTCACCAA 11761 ATCCATTATT ATCTGGATTA TTCACAAAT CAATGATAGA AGAAGATGAA GAATTAGCTG 11821 AGTTCCTGAT GGACAGGAAG G AATTC CC CTAGAGTTGC ACATGATATT CTAGATAATT 11881 CTCTCACAGG AATTAGAAAT GCCATAGCTG GAATGTTAGA TACGACAAAA TCACTAATTC 11941 GGGTTGGCAT AAATAGAGGA GGACTGACAT ATAGTTTGTT GAGGAAAATC AGTAATTACG 12001 ATCTAGTACA A7ATGAAACA CTAAGTAGGA CTTTGCGACT AATTGTAAGT GATAAAATCA 12061 AGTATGAAGA TATGTGTTCG GTAQACC TG CCATAGCATT GCGACAAAAG ATGTGGATTC 12121 ATTTA CAGG AGGAAGGATG ATAAGTGGAC TTGAAACGCC TGACCCATTA GAATTACTAT 12181 CTGGGGTAGT AATAACAGGA TCASAACATT GTAAAATATG TTATTCTTCA GATGGCACAA 12241 ACCCATATAC TTGGATGTAT TTACCCGGTA ATATCAAAAT AGCATCAGCA GAAACAGGTA 12301 TATCGTCATT AAGAGTTCCT TATTTTGGAT CAGTCACTGA TGAAAGATCT GAAGCACAAT 12361 TAGGATATAT CAAGAATCTT AGTAAACCTG CAAAASCCGC AATAAGAATA GCAATGATAT 12421 A7ACATGGGC ATTTGGTAAT GATGAGATAT CTTGGATGGA AGCCTCACAG ATAGCACAAA 12481 CACGTGCAAA TTTTACACTA GATAGTCTCA AAATTTTAAC ACCGGTAGCT ACATCAACAA 12541 ATTTATCACA CAGATTAAAG GATA CTGCAA CTCAGATGAA ATTCTCCAGT ACATCATTGA 12601 TCA6AGTCA6 CAGATTCATA ACAATGTCCA ATGATAACAT GTCTATCAAA GAAGCTAATG 12661 AAACCAAAGA TACTAATCTT ATTTATCAAC AAATAATG T AACAGGATTA A6TGTTTTCG 12721 AATATTTATT TAGATTAAAA GAAACCACAG GACACAACCC TATAGTTATG CATCTGCACA 12781 TAGAAGATGA GTGTT6TATT AAAGAAAGTT TTAATGATGA ACATATTAAT CCAGAGTCTA 12841 CATTAGAATT AATTCGATAT CCT5AAAGTA ATGAATTTAT TTATGATAAA GACCCACTCA 12901 AAGATGTGGA CTTATCAAAA CTTATGGTTA TTAAAGACCA TTCTTACACA ATTGATATGA 12961 ATTATTGGGA TGATACTGAC ATCATACATG CAATTTCAAT ATGTACTGCA ATTACAATAG_13021_CAGATACTAT GTCACAATTA GATCGAGATA ATTTAAAAGA GATAATAGTT ATTGCAAATG 13081 ATGATGATAT TAATAGCTTA ATCACTGAAT TTTTGACTCT TGACATACTT GTATTTCTCA 13141 AGACATTTGG TGGATTATTA GTAAATCAAT TTGCATACAC TCTTTATAGT CTAAAAATAG_13201_AAGGTAGGGA TCTCATTTGG GATTATATAA- TGAGAACACT GAGAGATACT TCCCATTCAA 13261 TATTAAAAGT ATTATCTAAT GCATTATCTC ATCCTAAAGT ATTCAAGAGG TTCTGGGATT 1332 GTGGAGTTTT AAACCCTATT TATG6TCCTA ATACTGCTAG TCAAGACCAG ATAAAACTTG 13381 CCCTATCTAT ATGTGAA TAT TCACTAGATC TATTTATGAG AGAATGGTTG AATGGTGTAT 13441 CACTTGAAAT ATACATTTGT GACAGCGATA TGGAAGTTGC AAATGATAGG AAACAAGCCT 13501 ttat TCTAG ACACCTTTCA TTTGTTTGTT GTTTAGCAGA AATTGCATCT TTCGGACCTA 13561 ACCTGTTAAA CTTAACATAC TTGGAGAGAC TTGATCTATT GAAACAATAT CTTGAATTAA 13621 ATATTAAAGA AGACCCTACT CTTAAATATG TACAAATATC TGGATTATTA ATTAAATCGT 13681 TCCCATCAAC TGTAACATAC GTAAGAAAGA CTGCAATCAA ATATCTAAGG ATTCGCGGTA 13741 TTAGTCCACC TGAGGTAATT GATGATTGGG ATCCGGTAGA AGATGAAAAT ATGCTGGATA 13801 ACATTGTCAA AACTATAAAT GATAACTGTA ATAAAGATAA TAAAGGGAAT AAAATTAACA 13861 ATTTCTGGGG ACTAGCACTT AAGAACTATC AAGTCCTTAA AATCAGATCT ATAACAAGTG 13921 ATTCTGATGA TAATGATAGA CTAGATGCTA ATACAAGTGG TTTGACACTT CCTCAAGGAG 13981 GGAATTATCT ATCGCATCAA TTGAGATTAT TCG5AATCAA CAGCACTAGT TGTCTGAAA6 14041 CTCTTGAGTT ATCACAAATT TTAATGAAGG AAGTCAATAA AGACAAGGAC AGGCTCTTCC 14101 TGGGAGAAGG AGCAGGAGCT ATGCTAGCAT GTTATGATGC CACATTAGGA CCTGCAGTTA 1161 ATTATTATAA TTCAGGTTTG AATATAACAG ATGTAATTGG TCAACGAGAA TTGAAAATAT 14221 TTCCTTCAGA GGTATCATTA GTAGGTAAAA AATTAGGAAA TGTGACACAG ATTCTTAACA 14281 GGGTAAAAGT ACTGTTCAAT GGGAATCCTA ATTCAACATG GATAGGAAAT ATGGAATGTG 14341 AGAGCTTAAT ATGGAGTGAA TTAAATGATA AGTOCATTGG ATTAGTACAT TGTGATATGG 14401 AAGGAGC AT CGGTAAATCA GAAGAAACTG TTCTACATGA ACATTATAGT GTTATAAGAA 14461 TTACATACTT GATTGGGGAT GATGATGTTG TTTTAGTTTC CAAAATTATA CCTACAATCA 14521 CTCCGAATTG GTCTAGAATA CTTTATCTAT ATAAATTATA TTGGAAAGAT GTAAGTATAA 14591 TATCACTCAA ACTTCTAAT CCTGCATCAA CAGAATTATA T TAATTTCG AAAGATGCAT 14641 ATTGTACTAT AATGGAACCT AGTGAAATTG T TATCAAA ACTTAAAAGA TTGTCACTCT 14701 TGGAAGAAAA TAATCTATTA AAATGGATCA TTTTATCAAA GAAGAGGAAT AATGAATGGT 14761 TACATCATGA AATCAAAGAA GGAGAAAGAG ATTATGGAAT CATGA6ACCA TATCATATGG 14821 CACTACAAAT CTTTGGATTT CAAATCAATT TAAATCATCT GGCGAAAGAA TTTTTATCAA 14881 CCC AGATCT GACTAATATC AACAATATAA TCCAAAGTTT TCAGCGAACA ATAAAGGATG 14941 TTTTATTTGA ATGGATTAAT ATAACTCATG ATGATAAGAG ACATAAATTA GGCGGAAGAT 15001 ATAACATATT CCCACTGAAA AATAAGGGAA AGTTAAGACT GCTATCGAGA AGACTAGTAT 15061 TAAG TTGGAT TTCATTATCA TTATCGACTC GATTACTTAC AGGTCGCTTT CCTGATGAAA 15121 AATTTGAACA TAGAGCACAG ACTGGATATG TATCATTAGC TGATACTGAT TTAGAATCAT 15181 TAAAGTTATT GTCGAAAAAC ATCATTAAGA ATTACAGAGA GTGTATAGGA TCAATATCAT 15241 ATTGGTTTCT AACCAAAGAA GTTAAAATAC TTATGAAATT GATCGGTGGT GCTAAATTAT 15301 TAGGAATTCC CAGACAATAT AAAGAACCCG AAGACCAGTT ATTAGAAAAC TACAATCAAC 15361 ATGATGAATT TGATATCGAT TAAAACATAA ATACAATGAA GATATATCCT AACCTTTATC 15421 TTTAAGCCTA GGAATASACA AAAAGTAAGA AAAACATGTA ATATATATAT ACCAAACAGA 154 BL GTTCTTCTCT TGTTTGGTstrategy (Figure 19), the PIV3-PIV2 F and HN genes were constructed in which the FIV and FN ORF regions of PIV2F coding for ectodomains and transmembrane domains were amplified from pLit. PIV32Fhc and pLit. PIV32HNhc, respectively, using PCR, Vent DNA polymerase and primer pairs specific for PIV2 F (13, 14 in Table 22) and PIV2 HN (15, 16 in Table 22). In parallel, the partial ORFs of the PIV3 F and HN genes encoding the ectodomains plus the transmembrane domains were deleted from their cDNA subclones pLit.PIV3.F3a and pLit.PIV3.HN4 (Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference), respectively, using PCR, Vent DNA polymerase, and primer pairs specific for PIV3 F (17, 18 in Table 22) and PIV3 HN (19). , 20 in Table 22). The F and HN cDNA fragments from PIV2 and PIV3 were gel purified and ligated to generate pLit. PIV32FCT and pLit. PIV32HNCT, respectively. These chimeric F and HN constructs were digested with PpuMI plus Spel and assembled together to generate pLit. PIV32CT, which was sequenced through the specific PIV region in its entirety and was found to be as designed. The 4 kb BspEI-Spel fragment from pLit.PIV32CT was introduced into the pEI-Spel window of p38'APIV31hc to generate p38'APIV32CT. The 6.5 kb BspEI-Sp l fragment from p38 'APIV32CT, containing the F and HN chimeric genes of PIV3-PIV2 was introduced into the BspEI-Sphl window of pFLC.2G + .hc and pFLCcp45, to generate pFLC.PIV32CT (Table 25, SEQ ID NO.62) and pFLC. PIV32CTcp45, respectively. The nucleotide sequence of this BspEl-Spel fragment was presented in GenBank.

TABLE 25 (SEQ ID NO 62) Sequence of pFLC. PIV32CT, 15474 base pairs in sense orientation (only the insert shown) 1 ACCAAACAAG AGAAGAAACT TGTCTGGGAA TATAAATTTA ACTTTAAATT AACTTAGGAT 61 TAAAGACATT GACTAGAAGG TCAAGAAAAG GGAACTCTAT AATTTCAAAA ATGTTGAGCC 121 TATTT6ATAC ATTTAATGCA CGTA6GCAA6 AAAACATAAC AAAATCAGCC GGTGGAGCTA 181 TCATTCCTGG ACAGAAAAAT ACTGTCTCTA TATTCGCC T TGGACCGACA ATAACTGATG 241 ATAATGAGAA AATGACATTA GCTCTTCTAT TTCTATCTCA TTCACTAGAT AATGAGAAAC 301 AACATGCACA AAGGGCAGGG TTCTTGGTGT CTTTATTGTC AATGGCTTAT GCCAATCCAG 361 AGCTCTACCT AACAACAAAT GGAAGTAATG CAGATGTCAA GTATGTCATA TACATGATTG 421 AGAAAGATCT AAAACGGCAA AAGTATGGAG GATTTGTGGT TAAGACGAGA GAGATGATAT 481 ATGAAAAGAC AACTGATTGG ATATTTGGAA GTGACCTGGA TTATGATCAG GAAACTATGT 541 TGCACAACGG CAGGAACAAT TCAACAA TG AAGACCTTGT CCACACATTT 6G6TATCCAT 601 CATGTTTAGG AGCTCTTATA ATACAGATCT GGATAGTTCT GGTCAAAGCT ATCACTA6TA 661 TCTCAGGGTT AAGAAAAGGC TTTTTCACCC GATTGGAAG TTTCAGACAA GATGGAACAG 721 TGCAGGCAGG GCTGGTATTG AGCGGTGACA CAGTGGATCA GATTGGGTCA ATCATGCGGT 78 1 CTCAACAGAG CTTGGTAACT CTTAT6GTTG AAACATTAAT AACAATGAAT ACCAGCAGAA B41 ATGACCTCAC AACCATAGAA AAGAATATAC AAATTGTTGG CAACTACATA AGAGATGCAG 901 GTCTCGCTTC ATTCTTCAAT ACAATCAGAT ATGGAATTGA GACCAGAATG GCAGCTTTGA 961 CTCTATCCAC TCTCAGACCA GATATCAATA GATTAAAAGC TTTGATGGAA CTGTATTTAT 1021 CAAAGGGACC ACGCGC OCT TTCATCTGTA TCCTCAGAGA TCCTATACAT GGTGAGTTCG 1081 CACCAGGCAA CTATOC GOC ATATGGAGCT ATGCAATGGG GGTGGCAGTT GTAdAAAATA 1141 GAGCCATGCA ACAGTATGTG ACGGGAAGAT CATATCTAGA CATTGATATG TTCCAGCTAG_1201_GACAAGCAGT AGCACGTGAT GCCGAAGCTC AAATGAGCTC AAGACTGGAA GATGAACTTG 1261 GAGTGACACA CGAATC AAA GAAAGCTTGA AGAGACATAT AAGGAACATA AACAGTTCAG 1321 AGACATCTTT CCACAAACCG ACAGGTGGAT CAGCCATAGA GATGGCAATA GATGAAGAGC 1381 CAGAACAATT CGAACATAGA GCAGAXCAAG AACAAAATGG A6AACCTCAA TCATCCATAA 144 TTCAATATGC CTGGGCAGAA GGAAATAGAA GCGATGATCA GACTGAGCAA GCTACAGAAT 1501 CTGACAATAT CAAGACCGAA CAACAAAACA TCAGAGACAG ACTAAACAAG AGACTCAACG 1561 ACAAGAAGAA ACAAAGCAGT CAACCACCCA CTAATCCCAC AAACAGAACA AACCAGGACG 621 AAATAGATG A TCTGTTTAAC GCATTTGGAA GCAACTAATC GAATCAACAT TTTAATCTAA 1681 ATCAATAATA AATAAGAAAA ACTTAGGATT AAAGAATCC ATCATACCGG AATATAGGGT 1741 GGTAAATTTA GAGTCTGCTT GAAACTCAAT CAATAGAGAG TTGATGGAAA GCGATGC AA 1801 AAACTATCAA ATCATGGATT CTTGGGAAGA GGAATCAAGA GATAAATCAA CTAATATCTC 1861 CTCGGCCCTC AACATCATTG AATTCATACT CAGCACCGAC CCCCAAGAAG ACTTATCGGA 1921 AAACGACACA ATCAACACAA GAACCCAGCA ACTCAGTGCC ACCATCIGTC AACCAGAAAT 1981 CAAACCAACA GAAACAAG G AGAAAGATAG TGGATCAACT GACAAAAATA GACAGTCCGG 2041 GTCATCACAC GAATGTACAA CAGAAGCAAA 'AGATAGAAAT ATTGATCAGG AAACTGTACA 2101 GAGAGGACCT GGGAGAAGAA GCAGCTCAGA TAGTAGAGCT GAGACTGTGG TCTCTGGAGG 2161 AATCCCCAGA AGCATCACAG. ATTCTAAAAA TGGAACCCAA AACAOSGAGG ATATTGATCT 2221 CAATGAAATT AGAAAGATGG ATAAGGACTC TATTGAGGGG AAAATGCGAC AATCTGCAAA 2281 TGTTCCAAGC GAGATATCAG GAAGTGATGA CATATTTACA ACAGAACAAA GTAGAAACAG 2341 TGATCATG6A AGAAGCCTGG AATCTATCAG TACACCTGAT ACAAGATCAA TAAGTGTTGT 2401 TACTGCTGCA ACACCAGATG ATGAAGAAGA AATACTAATG AAAAATAGTA GGACAAAGAA 2461 AAGTTCTTCA ACACATCAAG AAGATGACAA AAGAATTAAA AAAGGGGGAA AAGGGAAAGA 2321 CTGGTTTAAG AAATCAAAAG ATACCGACAA CCAGATACCA ACATCAGACT ACAGATCCAC 2581 ATCAAAAGGG CAGAAGAAAA TCTCAAAGAC AACAACCACC AACACCGACA CAAAGGGGCA 2641 AACAGAAATA CAGACAGAAT CATCAGAAAC ACAATCCTCA TCATGGAATC TCATCATCGA 2701 CAAAACACC GACCGGAACG AACAGACAAG CACAACTCCT CCAACAACAA CTTCCAGATC 2761 AACTTATACA AAAGAATCGA TCCGAACAAA CTCTGAATCC AAACCCAAGA CACAAAAGAC 2821 AAATGGAAAG GAAAGGAAGG ATACAGAAGA GAGCAATCGA TTTACAGAGA GGGCAATTAC 2881 TCTATTGCAG AATCTTGGTG TAATTCAATC CACATCAAAA CTAGATTTAT ATCAAGACAA 2941 ACGAGTTGTA TGTGTAGCAA ATGTACTAAA CAA GTAGAT ACTGCATCAA AGATAGATTT 300 CCTGGCAGGA TTAGTCATAG GGGTTTCA AT GGACAACGAC ACAAAATTAA CACAGATACA 3061 AAATGAAATG CTAAACCTCA AAGCAGATCT AAAGAAAATG GACGAATCAC ATAGAAGATT 3121 GATAGAAAAT CAAAGAGAAC AACTGTCATT GATCACGTCA CTAATTTCAA ATCTCAAAAT 3191 TATGACTGAG AGAGGAGGAA AGAAAGACCA AAATGAATCC AATGAGAGAG TATCCATGAT 3241 CAAAACAAAA TTGAAAGAAG AAAAGATCAA GAAGACCAGG TTTGACCCAC TTATGGAGGC 3301 ACAAGGCATT GACAAGAATA TACCCGATCT ATATCGACAT GCAGGAGATA CACTAGAGAA 3361 CGATGTACAA GTTAAATCAG AGATATTAAG TTCATACAAT GAGTCAAATG CAACAAGACT 3421 AATACCCAAA AAAGTGAGCA GTACAATGAG ATCACTAGTT GCAGTCATCA ACAACAGCAA 3481 TCTCTCACAA AGCACAAAAC AATCATACAT AAACGAACTC AAACGTTGCA AAAATGATGA 3S41 AGAAGTATCT GAATTAATGG ACATGTTCAA TGAAGATGTC AACAATTGCC AATGATCCAA 3601 CAAAGAAACG ACACCGAACA AACAGACAAG AAACAACAGT AGATCAAAAC CTG CAACAC 3661 ACACAAAATC AAGCAGAATG AAACAACAGA TATCAATCAA TATACAAATA AGAAAAACTT 3721 AGGATTAAAG AATAAATTAA TCCTTGTCCA AAATGAGTAT AACTAACTCT GCAATATACA 3781 CATTCCCAGA ATCATCATTC TCT6AAAAT6 GTCATATAGA ACCATTACCA CTCAAAGTCA 3841 ATGAACAGAG GAAAGCAGTA CCCCACATTA GAG TTGCCAA GATCGGAAAT CCACCAAAAC 3901 ACGGATCCCG GTATTTAGAT GTCTTCTTAC TCGGCTTCTT CGAGATGGAA CGAATCAAAG 3961 ACAAATACGG GAGTGTGAAT GATCTCGACA GTGACCCGAG TTACAAAGTT TGTGGCTCTG 4021 GATCATTACC AATCGGATTG GCTAAGTACA CTGGGAATGA CCAGGAATTG TTACAAGCCG 4081 CAACCAAACT GGATATAGAA GTGAGAAGAA CAGTCAAAGC GAAAGAGATG GTTGTTTACA 4141 CGGTACAAAA TATAAAACCA GAACTGTACC CATGGTCCAA TAGACTAAGA AAAGGAATGC 4201 TGTTCGATGC CAACAAAGTT GCTCTTGCTC CTCAATGTCT TCCACTAGAT AGGAGCATAA 4261 AATTTAGAGT AATCTTCGTG AATTGTACGG CAATTGGATC AATAACCTTG TTCAAAATTC 4321 CTAAGTCAAT GGCATCACTA TCTCTACCCA ACACAATATC AATCAATCTG CAGGTACACA 4381 TAAAAACAGG GGTTCAGACT GATTCTAAAG GGATAGTTCA AATTTTGGAT GAGAAAGGCG 444 AAAAATCACT GAATTTCATG GTCCATCTCG GATTGATCAA AAGAAAAGTA GGCAGAATGT 4501 ACTCTGTTGA ATACTGTAAA CAGAAAATCG AGAAAATGAG ATTGATATTT TCTTTAGGAC 561 TAGTTGGAGG AATCAGTCTT CATGTCAATG CAACTGGGTC CATATCAAAA ACACTAGCAA 4621 GTCAGCTGGT ATTCAAAAGA GAGATTTGTT ATCCTTTAAT GGATCTAAAT CCGCATCTCA 4681 ATCTAGTTAT CTGGGCTTCA TCAGTAGAGA TTACAAGAGT GGATGCAATT TTCCAACCTT 4741 CTTTACCTGG CGAGTTCAGA TACTATCCTA ATATTATTGC AAAAGGAGTT GGGAAAATCA 4801 AACAATGGAA CTAGTAATCT CTATTTTAGT CCGGACGTAT CTATTAAGCC GAAGCAAATA 4861 AAGGATAATC AAAAACTTAG GACAAAAGAG GTCAATACCA ACAACTATTA GCAGTCACAC 4921 TCGCAAGAAT AAGAGAGAAG GGACCAAAAA AGTCAAATAG GAGAAATCAA AACAAAAGGT 4981 ACAGAACACC AGAACAACAA AATCAAAACA TCCAACTCAC TCAAAACAAA AATTCCAAA 5041 GAGACCGGCA ACACAACAAG CACTGAACAT GCATCACCTG CATCCAATGA TAGTATGCAT S101 TTTTGTTATG TACACTGGAA TTGTAGGTTC ASATGCCATT GCTGGAGATC AACTCCTCAA S161 TGTAGGGGTC ATTCAATCAAACTCATGTAC TACACTGATG GTGGCGCTAG_5221_CTTTATTGTT GTAAAATTAC TACCCAATCT TCCCCCAAGC AATGGAACAT GCAACATCAC 5281 CAGTCTAGAT GCATATAATG TTACCCTATT TAAGTTGCTA ACACCCCTGA TTGAGAACCT 53 1 GAGCAAAATT TCTGCTGTTÁ CAGATACCAA ACCCCGCCGA GAACGATTTG CAGGAGTCGT 5401 TATTGGGCrr GCTGCACTAG GAGTAGCTAC AGCTGCACAA ATAACCGCAG CtGTAGCAAT 5461 AGTAAAAGCC AATGCAAATG CT6CTGCGAT AAACAATCTT GCATCTTCAA TTCAATCCAC 5521 CAACAAGGCA GTATCCGATG TGATAACTGC ATCAAGAACA ATTGCAACCG CAGTTCAAGC 5581 GATTCAGGAT CACATCAATG GAGCCATTGT CAACGGGATA ACATCTGCAT CATGCCGTGC 5641 CCATGATGCA CTAATTGGGT CAATATTAAA TTTGTATCTC ACTGAGCTTA CTACAATATT 5701 TCATAATCAA ATAACAAACC CTGCGCTGAC ACCACTTTCC ATCCAAGCTT TAAGAATCCT 5761 CCTCGGTAGC ACCTTGCCAA TTGTCATTGA ATCCAAACTC AACACAAAAC TCAACACAGC 5821 AGAGCTGCTC AGTAGCGGAC TGTTAACTGG TCAAATAATT TCCATTTCCC CAATGTACAT 5881 GCAAATGCTA ATTCAAATCA ATGTTCCGAC ATTTATAATG CAACCCGGTG CGAAGGTAAT 5941 TGATCTAATT GC ATCTCTG CAAACCATAA ATTACAAGAA GTAGTTGTAC AAGTTCCTAA 6001 TAGAATTCTA GAATATGCAA ATGAA CTACA AAACTACCCA GCCAATGATT GTTTCGTGAC 6061 ACCAAACTCT GTATTTTGTA GATACAATGA GGGTTCCCCG ATCCCTGAAT CACAATATCA 6121 ATGCTTAAGG GGGAATCTTA ATTCTTGCAC TTTTACCCCT ATTATCGGGA ACTTTCTCAA 6181 GCGATTCGCA TTTGCCAATG GTGTGCTCTA TGCCAACTGC AAATCTTTGC TATGTAAGTG 624 TGCCGACCCT CCCCATGTTG TGTCTCAAGA 7GACAA CAA GGCATCAGCA TAATTGATAT 6301 TAAGAGGTGC TCTGAGATGA TGCTTGACAC TTTTTCATTT AGGATCACAT CTACATTCAA 6361 TGCTACATAC GTGACAGACT TCTCAATGAT TAATGCAAAT ATTGTACATC TAAGTCCTCT 6421 AGACTTGTCA AATCAAATCA ATTCAATAAA CAAATCTCTT AAAAGTGCTG AGSATTGGAT 6481 TGCAGATAGC AACTTCTTCG CTAATCAAGC CAGAACAGCC AAGACACTTT ATTCACTAAG 6541 TGCAATCGCA TTAATACTAT CAGTGATTAC TTTGGTTGTT GTGGGATTGC TGATTGCCTA 6601 CATCATCAAG TATTACAGAA TTCAAAAGAG AAATCGAGTG GATCAAAATG ACAAGCCATA 6661 TGTACTAACA AACAAATAAC ATATCTACAG ATCATTAGAT ATTAAAATTA TAAAAAACTT 6721 AGGAGTAAAG TTACGCAATC CAACTCTACT CATATAATTG AGGAAGGACC CAATAGACAA 6781 ATCCAAATTC GAGATGGAAT ACTGGAAGCA TACCAATCAC GGAAAGGATG CTGGTAATGA 6841 GCTGGAGACG TCTATGGCTA CTCATGGCAA C AAGCTCACT AATAAGACTG CCACAATTCT 6901 TGGCATATGC ACATTAATTG TGCTATGTTC AAGTATTCTT CATGAGATAA TTCATCTTGA 6961 TGTTTCCTCT GGTCTTATGA ATTCTGATGA GTCACAGCAA GGCATTATTC AGCCTATCAT 7021 AGAATCATTA AAATCATTGA TTGCTTTGGC CAACCAGATT CTATATAATG TTGCAATAGT 7081 AATTCCTCTT AAAATTGACA G ATCGAAAC TGTAATACTC T TGCTTTAA AAGATATGCA 4 CACCGGGAGT ATGTCCAATG CCAACTGCAC GCCAGGAAAT CTGCTTCTGC ATGATGCAGC 7201 ATACATCAAT GGAATAAACA AATTCCTTGT ACTTGAATCA TACAATGGGA CGCCTAAATA 7261 TGGACCTCTC CTAAATATAC CCAGCTTTAT CCCCTCAGCA ACATCTCCCC ATGGGTGTAC 7321 TAGAATACCA TCATTTTCAC TCATCAAGAC CCATTGGTGT TACACTCACA ATGTAATGCT 7381 TGGAGATTGT CTTGATTTCA CGGCATCTAA CCAGTATTTA TCAATGGGGA TAATACAACA 74 1 ATCTGCTGCA GGGTTTCCAA TTTTCAGGAC TATGAAAACC ATTTACCTAA GTGATG6AAT 7501 CAATCGCAAA AGCTGTTCAG TCACTGCTAT ACCAGGAGGT TGTGTCTTGT ATTGCTATGT 7561 AGCTACAAGG TCTGAAAAAG AAGATTATGC CACGACTGAT CTAGCTGAAC TGAGACTTGC 7621 TTTCTATTAT TATAATGATA CCTTTATTGA AAGAGTCATA TCTCTTCCAA ATACAACAGG 7681 GCAGTGGGCC ACAATCAACC CTGCAGTCGG AAGCGGGAT C TATCATCTAG GCTTTATCTT 774 ATTTCCTGTA TATGGTGGTC TCATAAATGG GACTACTTCT TACAATGAGC AGTCCTCACG 7801 CTATTTTATC CCAAAACATC CCAACATAAC TTGTGCCGGT AACTCCAGCA AACAGGCTGC 7861 AATAGCACGG AGTTCCTATG TCATCCGTTA TCACTCAAAC AGGTTAATTC AGAGTGCTGT 7921 TCTTATTTGT CCATTGTCTG ACATGCATAC AGAAGAGTGT AATCTAGTTA TGTTTAACAA July 81 TTCCCAAGTC ATGATGGGTG CAGAAGGTAG GCTCTATGTT ATTGGTAATA ATTTGTATTA 8041 TTATCAACGC AGTTCCTCTT GGTGGTCTGC ATCGCTCTTT TACAGGATCA ATACAGATTT 8101 TTCTAAAGGA ATTCCTCCGA TCATTGAGGC TCAATGGGTA CCGTCCTATC AAGTTCCTCG 8161 TCCTGGAGTC ATGCCATGCA ATGCAACAAG TTTTTGCCCT GCTAATTGCA TCACAGGGGT 8221 GTACGCASAT GTGTGGCCGC TTAATGATCC AGAACTCATG TCACGTAATG CTCTGAACCC 8281 CAACTATCGA TTTGCTGGAG CCTTTCTCAA AAATGAGTGC AACCGAACTA ATCCCACATT 8341 CTACACTGCA TCGGCTAACT CCCTCTTAAA TACTACCGGA TTCAACAACA CCAATCACAA 8401 AGCAGCATAT ACATCTTCAA CCTGCTTTAA AAACACTGGA ACCCAAAAAA TTTATTGTTT 6461 AATAATAATT GAAATG6GCT CATCTCTTTT AGGGGAGTTC CAAATAATAC CATTTTTAAG 8S21 GGAACTAATG CTTTAATCAT AATTAACCAT AATATGCATC AATCT ATCTA8581 ATATGATAAG TAATCAGCAA TCAGAC TA GACAAAAGGG AAATATAAAA AACTTAGGAG 8641 CAAAGCGTGC TCGGGAAATG GACACTGAAT CTAACAATGG CACTGTATCT GACATACTCT 8701 ATCCTGAGTG TCACCTTAAC TCTCCTATCG TTAAAGGTAA AATAGCACAA TTACACACTA 8761 TTATGAGTCT ACCTCAGCCT TATGATATGG ATGACGACTC AATACTAGTT ATCACTAGAC 8821 AGAAAATAAA ACTTAATAAA TTGGATAAAA GACAACGATC TATTAGAAGA TTAAAATTAA 8881 TATTAACTGA AAAAGTGAAT GACTTAGQAA AATACACKXT TATCAGATAT CCAGAAATGT 8941 CAAAAGAAAT GTTCAAATTA TATATACCTG GTATTAACAG TAAAGTGACT GAATTATTAC 9001 TTAAAGCAGA TAGAACATAT AGTCAAATGA CTGATGGATT AAGAGATCTA TGGATTAATG 9061 TGCTATCAAA ATTAGCCTCA AAAAATGATG GAAGCAATTA TGATCTTAAT GAAGAAATTA 9121 ATAATATATC GAAAGTTCAC ACAACCTATA AATCAGATAA ATGGTATAAT CCATTCAAAA 9181 CATGGTTTAC TATCAAGTAT GATATGAGAA GATTACAAAA AGCTCGAAAT GAGATCACTT 9241 TTAATGTTGG GAAGGATTAT AACTTGTTAG AAGACCAGAA GAATTTCTTA TTGATACATC 9301 CAGAATTGGT TTTGATATTA GATAAACAAA ACTATAATGG TTATCTAATT ACTCCTGAAT 9361 TAGTATTGAT GTATTGTGAC GTAGTCGAAG GCCGATGGAA TATAAGTGCA TGTGCTA AGT 9421 TAGATCCAAA ATTACAATCT ATGTATCAGA AAGGTAATAA C TGTGGGAA GTGATAGATA 9481 AATTGTTTCC AATTATGGGA GAAAAGACAT TTGATGTGAT ATCGTTATTA GAACCACTTG 9541 CATTATCCTT AATTCAAACT CATGATCCTG TTAAACAACT AAGAGGAGCT TTTTTAAATC 9601 ATGTGTTATC CGAGATGGAA TTAATATTTG AATCTAGAGA ATCGATTAAG GAATTTCTGA 9661 GTGTAGATTA CATTGATAAA ATTTTAGATA TATTTAATAA GTCTACAATA GATGAAATAG_9721_CAGAGATTTT CTCTTTTTTT AGAACATTTG GGCATCCTCC ATTAGAAGCT AG ATTGCAG 9781 CAGAAAAGGT TAGAAAATAT ATGTATATTG GAAAACAATT AAAATTTGAC ACTATTAATA 9841 AATGTCATGC TATCTTCTGT ACAATAATAA TTAACGGATA TAGAGAGAGG CATGGTGGAC 9901 AGTGGCCTCC TGTGACATTA CCTGATCATG CACACGAATT CATCATAAAT GCTTACGGTT 9961 CAAACTCTGC GATATCATAT GAAAATGCTG TTGATTATTA CCAGAGCTTT ATAGGAATAA 10021 AATTCAATAA ATTCATAGAG CCTCAGTTAG ATGAGGATTT GACAATTTAT ATGAAAGATA 10,081 AAGCATTATC TCCAAAAAAA TCAAATTGGG ACACAGTTTA TCCTGCATCT AATTTACTGT 10141 ACCGTACTAA CGCATCCAAC GAATCACGAA GATTAGTTGA AGTATTTATA GCAGATAGTA 10201 AATTTGATCC TCATCAGATA TTGGATTATG TAGAATCTGG GGACTGGTTA GATGATCC AG 10261 AATTTAATAT TTCTTATAGT CTTAAAGAAA AAGAGATCAA ACAGGAAGGT AGACTCTTTG 10321 CAAAAATGAC ATACAAAATG AGASCTACAC AAGTTTTATC AGAGACACTA CTTGCAAATA 10381 ACATAGGAAA ATTCTTTCAA GAAAATGGGA TGGTGAAGGG AGAGATTGAA TTACTTAAGA 10441 GATTAACAAC CATATCAATA TCAGGAGTTC CACGGTATAA TGAAGTGTAC AATAATTCTA 10501 AAAGCCATAC AGATGACCTT AAAACCTACA ATAAAATAAG TAATCTTAAT TTGTCTTCTA 10561 ATCAGAAATC AAAGAAATTT GAATTCAAGT CAACGGATAT CTACAATGAT GGATACGAGA 10621 CTGTGAGCTG TTTCCTAACA ACAGATCTCA AAAAATACTG TCTTAATTGG AGATATGAAT 10681 CAACAGCTCT ATTTGGAGAA ACTTGCAACC AAATATTTGG ATTAAATAAA TTGTTTAATT 10741 GGTTACACCC TCGTCTTGAA GGAAGTACAA TCTATGTAGG TGATCCTTAC TGTCCTCCAT 10801 CAGATAAAGA ACATATATCA TTAGAGGATC ACCCTGATTC 'TGGTTTTTAC GTTCATAACC 10861 CAAGAGGGGG TATAGAAGGA TTTTGTCAAA AATTATGGAC ACTCATATCT ATAA6TGCAA 10921 TACATCTAGC AGCTGTTAGA ATAGGCGTGA GGGTGACTGC AATGGTTCAA GGAGACAATC 10981 AAGCTATAGC TGTAACCACA AGAGTACCCA ACAATTATGA CTACAGAGTT AAGAASGAGA 110 1 TAG TATAA AGATGTAGTG AGATTTTTTG ATTCATTAAG A6AAGTGATG 6AT6ATCTA6 11101 GTCAT6AACT TAAATTAAAT GAAACGATTA TAAGTA6CAA GATGTTCATA TATAGCAAAA 11161 GAATCTATTA TGATGGGAGA ATTCTTCCTC AAGCTCTAAA AGCATTATCT AGATGTGTCT 11221 TCTGGTCAGA GACAGTAATA GACGAAACAA GATCAGCATC TTCAAATTT6 GCAACATCAT 11281 TTGCAAAAGC AATTGAGAAT GGTTATTCAC CTGTTCTAG_6_ATATGCATGC TCAATTTTTA 11341 AGAATATTCA ACAACTATAT ATTGCCCTTG GGATGAATAT CAATCCAACT ATAACACAGA 11401 ATATCASAGA TCAGTATTTT AGGAATCCAA ATTGGATGCA ATATGCCTCT TTAATACCTG 11461 CTAGTGTTGG GGGATTCAAX TACATGGCCA TGTCAAGATG TTTTGTAAGG AATAT GGTG 11521 ATCCATCAGT TGCCGCATTG GC GATATTA AAAGATTTAT TAAGGCGAAT CTATTAGACC 11581 GAAGTGTTCT TTATAGGATT ATGAATCAAG AACCAGGTGA GTCATCTTTT TTGGACTGGG 11641 CTTCAGATCC ATATTCATGC AATTTACCAC AATCTCAAAA TATAACCACC ATGATAAAAA 1170 ATATAACAGC AAGGAATGTA TTACAAGATT CACCAAATCC ATTATTATCT GGATTATTCA 11761 CAAATACAAT GATAGAAGAA GATQAAGAAT TAGCTGAGTT CCTGATGGAC AGGAAGGTAA 11821 TTCTCCCTAG AGTTGCACAT GATATTCTAG ATAATTCTCT CACAGGAATT AGAAATGCCA 11881 TAGCTGGAAT GTTAGATACG ACAAAATCAC TAATTOGGGT TGG CATAAAT11941 TGACATATAG TTTGTGAGG AAAATCAGTA ATTACGATCT AGTACAATAT GAAACACTAA 12001 GTAGGACTTT GCGACTAATT GTAAGTGATA AAATCAAGTA TGAAGATATG TGTTCGGTAG ACCTTGCCAT AGCATTGCGA.CAAAAGATGT 12061 12121 GGATTCATTT ATCAGGAGGA AGGATGATAA GTGGACTTGA AACGCCTGAC CCATTAGAAT TACTATCTGG GGTAGTAATA ACAGGATCAG 12181 AACATTGTAA AATATGTTAT TCTTCAGATG GCACAAACCC ATATACTTGG AT6TATTTAC 12241 CCGGTAATAT CAAAATAGGA TCAGCAGAAA CASGTATATC GTCATTAAGA GTTCCTTATT 12301 TTGGATCAG CACTGATGAA AGATCTGAAG CACAATTAGG ATATATCAAG AATCTTAGTA. 12361 AACCTGCAAA AGCCGCAATA AGAATAGCAA TGATATATAC ATGGGCATTT GGAATGATG 12421 AGATATCTTG GATGGAAGCC TCACAGATAG CACAAACACG TGCAAATTTT ACACTAGATA 12481 GTCTCAAAAT TTTAACACCG GTAGCTACAT CAACAAATTT ATCACACAGA TTAAAGGATA 12541 CTGCAACTCA GATGAAATTC TCCAGTACAT CATTGATCAG AGTCAGCAGA TTCATAACAA 12601 TGTCCAATGA TAACATGTCT ATCAAAGAAG CTAATGAAAC CAAAGATACT AATCTTATTT 12661 ATCAACAAAT AATGTTAACA GGATTAAGTG TTTTCGAATA TTTATTTAGA TTAAAAGAAA 12721 CCACAGGACA CAACCCTATA GTTATGCATC TGCACATAGA AGATGAGTGT TGTATTAAAG 12781 AAAGTTTTAA TGATGAACAT ATTAATCCAG AGTCTACATT AGAATTAATT CGATATCCTG 12841 AAAGTAATGA ATTTATTTAT GATAAAGACC CACTCAAAGA TGTGGACTTA TCAAAACTTA 12901 TGGTTATTAA AGACCATTCT TACACAATTG ATATGAATTA TTGGGATGAT ACTGACATCA 12961 TACATGCAAT TTCAATATGT ACTGCAATTA CAATAGCAGA TACTATGTCA CAATTAGATC 13021 GAGATAATTT AAAAGAGATA ATAGTTATTG CAAATGATGA TGATATTAAT AGCTTAATCA 13081 CTGAATTTTT GACTCTGAC ATACTTGTAT TTCTCAAGAC ATTTGGTGGA TTATTAGTAA 13141 ATCAATTTGC ATACACTCTT TATAGTCTAA AAATAGAAGG TAGGGATCTC ATTTG GGATT 13201 ATATAATGAG AACACTGAGA GATACTTCCC ATTCAATATT AAAAGTATTA TCTAATGCAT 13261 TATCTCATCC TAAAGTATTC AAGAGGTTCT GGGATTGTGG AGTTTTAAAC CCTATTTATG 13321 GTCCTAATAC TGCTAGTCAA GACCAGATAA AACTTGCCCT ATCTATATGT GAATATTCAC 13381 TAGATCTATT TATGAGAGAA TGGTTGAATG GTGTATCACT TGAAATATAC ATTTGTGACA 134 1 GCGATATGGA AGTTGCAAAT GAIAGGAAAC AAGCCTTTAT TTCTAGACAC CTTTCATTTG 13S01 TTTGTTGTTT AGCAGAAATT GCATCTTTCG GACCTAACCT GTTAAACTTA ACATACTTGG 13561 AGAGACTTGA TCTATTGAAA CAATATCTTG AA TAAATAT TAAAGAAGAC CCTACTCTTA 13621 AATATGTACA AATATCTGGA ???? G ????? AATCGTTCCC ATCAACTGTA ACATACGTAA 13681 GAAAGACTGC AATCAAATAT CTAAGGATTC GCGGTATTAG TCCACCTGAG GTAATTGATG 13741 ATTGGGATCC GGTAGAAGAT GAAAATATGC TGGATAACAT TTCAAAACT ATAAATGATA 13801 ACTGTAATAA AGATAATAAA GGGAATAAAA TTAACAATTT CTGGGGACTA GCACTTAAGA 13861 ACTATCAAGT CCTTAAAATC AGATCTATAA CAAGTGATTC TGATGATAAT GATAGACTAG_13921_ATGCTAATAC AAGTGGTTTG ACACTTCCTC AAGGAGGGAA TTATCTATCG CATCAATTGA 13981 GATTATTCGG AATCAACAGC ACTAGTTGTC TGAAAGCTCT TGAGTTATCA CAAATTTTAA 14041 TGAAGGAAGT CAATAAAGAC AAGGACAGGC TCTTCCTGGG AGAAGGAGCA GGAGCTATGC 14101 TAGCATGTTA TGATGCCACA TTAGGACCTG CAGTTAATTA TTATAATTCA GGTTTGAATA 14161 TAACASAT6T AATTGGTCAA CGAGAATTGA AAATATTTCC TTCAGAGGTA TCATTAGTAG_14221_GTAAAAAATT AGGAAATGTG ACACAGATTC TTAACAGGGT AAAAGTACTG TTCAATGGGA 14281 ATCCTAATTC AACATGGATA GGAAATATGG AATGTGAGAG CTTAATATGG AGTGAATTAA 14341 ATGATAAGTC CATTGGATTA GTACATTGTG ATATGGAAGG AGCTATCGGT AAATCAGAAG 14401 AAACTGTTCT ACATGAACAT TATAGTGTTA TAAGAATTAC ATACTTGATT GGGGATGATG ATGTTGTTTT AGTTTCCAAA 14461 ATTA TACCTA CAATCACTCC GAATTGGTCT AGAATACTTT 14521 ATCTATATAA ATTATATTGG AAAGATGTAA GTATAATATC ACTCAAAACT TCTAATCCTG 14581 CATCAACAGA ATTATATCTA ATTTCGAAAG ATGCATATTG TACTATAATG GAACC AGTG 146 AAATTGTTTT ATCAAAACTT AAAAGATTGT CACTCTTGGA AGAAAATAAT CTATTAAAAT 14701 GGATCATTTT ATCAAAGAAG AGGAATAATG AATGGTTACA TCAT6AAATC AAAGAAGGAG 14761 AAAGAGATTA TGGAATCATG AGACCATATC ATATGGCACT ACAAATC T GGATTTCAAA 14821 TCAATTTAAA TCATCTGGCG AAAGAATTTT TATCAACCCC AGATCTGACT AATATCAACA 14 ere 1 ATATAATCCA AAGTTTTCAG CGAACAATAA AGGATGTTTT ATTTGAATGG ATTAATATAA 14941 CTCATGATGA TAAGAGACAT AAATTAGGCG GAAGATATAA CATATTGCCA CTGAAAAATA 15001 AGGGAAAGTT AAGACTGCTA TCGAGAAGAC TAGTATTAAG TTGGATTTCA TTATCATTAT 15061 CGACTCGATT ACTTACAGGT GGCTTTCCTG ATGAAAAATT TGAACATAGA GCACAGACTG 15121 GATATGTATC ATTAGCTGAT ACTGATTTAG AATCATTAAA GTTATTGTCG AAAAACATCA 15181 TTAAGAATTA CAGAGAGTGT ATAG_6_ATCAA TATCATATTG GTTTCTAACC AAAGAAGTTA 15241 AAATACTTAT GAAATTGATC GGTGGTGCTA AATTATTAGG AATTCCCAGA CAKTATAAAG 15301 AACCCGAAGA CCAGTTATTA GAAAACTACA ATCAACATGA TGAATTTGAT ATCGATTAAA 15361 ACATAAATAC AATGAAGATA TATCCTAACC TTTATCTTTA AGCCTAGGAA TAGACAAAAA 15421 GTAAGAAAAA CATGTAATAT ATATATACCA AACAGAGTTC TTCTCTTGTT TGGT The cDNA engineering was designed in such a way that the PIV3-2 antigenomes conformed to the six rule (Calain et al., J. Virol 67: 4822-30, 1993; Durbin et al., Virology 234: 74- 83, 1997, each incorporated herein by reference). The PIV3-2 insert in pFLC.PIV32TM is 15498 nt in length and that in pFLC.PIV32CT is 15474 nt in length. These total lengths do not include the two G '5 terminal residues contributed by the T7 promoter, because it was assumed that they were removed during recovery.

Transfection and recovery of recombinant chimeric PIV3-PIV2 viruses HEp-2 cell monolayers were developed for confluence in six-well plates, and transfections were performed practically as described (Tao et al., 72: 2955-2961, 1998, incorporated herein by reference). The HEp-2 monolayer in a cavity was transfected with 5 g of PIV3-PIV2 antigenomic cDNA and three support plasmids, 0.2 g pTM (N), 0.2 μg pTM (PnoC), 0.1 μg pTM (L) in 0.2 ml of MEM which contains 12 μ? of LipofectACE (Life Technologies). The cells were simultaneously infected with MV A-T7 at a multiplicity of infection (MOI) of 3 in 0.8 ml of serum-free MEM containing 50 g / ml of gentamicin and 2m glutamine. The chimeric antigenomic cDNA pFLC.2G + .hc (Tao et al., J. Virol., 72: 2955-2961, 1998) was transfected in parallel as a positive control. After incubation at 32 ° C for 12 hours, the transfection medium was replaced with 1.5 ml of ME free of presco serum supplemented with 50 μg ml of gentamicin and 2 mM glutamine. The transfected cells were incubated at 32 ° C for two additional days. Porcine trypsin irradiated by gamma (p-trypsin; T1311, Sigma, St Louis, MO) was added to a final concentration of 0.5 μg ml on day 3 after transfection. Cell culture supernatants were harvested and passaged (referred to as passage 1) on fresh Vero cell monolayers in T25 flasks. After overnight adsorption, the transfection collection was replaced with fresh VP-SFM supplemented with 0.5 μg ml of p-trpsin. The cultures from passage 1 were incubated at 32 ° C for 4 days and the amplified virus was collected and further passaged on Vero cells (designated as passage 2) for another 4 days at 32 ° C in the presence of 0.5 μg ml of p-trypsin. The presence of viruses in the cultures of passage 2 was determined by hemadsorption with 0.2% guinea pig erythrocytes (RBC). The viruses were further purified by three consecutive terminal dilutions performed using Vero cells that were maintained in VP-SFM supplemented with 2 mM glutamine, 50 μg ml of gentamicia and 0.5 g ml of p-trypsin. After the third terminal dilution, the virus was further amplified three times on Vero cells and this virus suspension was used for further characterization in vitro and in vivo.

Confirmation of the chimeric nature of vRNA utilizing sequencing and restriction analysis of PCR products For the analysis of the genetic structure of the vRNAs, the recombinant PIVs were amplified in LLC-MK2 cells and concentrated. The vRNA was extracted from the viral granules and reverse transcribed using the Superscript Preamplification system. RT-PCR was performed using the Advantage cDNA synthesis kit and primer pairs specific for PIV2 or PIV3 cDNA (21, 22 or 23, 24 in Table 22). The RT-PCR products were either analyzed by restriction digestion or gel purified and analyzed by sequencing.

Replication of PIV in LLC-MK2 cells The development of PIV viruses in tissue culture was evaluated by infecting confluent LLC-MK2 cell monolayers in six-well plates in triplicate at an MOI of 0.01. The inoculum was removed after absorption for 1 hour at 32 ° C. The cells were washed 3 times with OptiMEM I, free of serum, were fed with 2 ml / cavity of OptiMEM I supplemented with 50 μ? /? a? of gentamicin and 0.5 μg ml of p-trypsin, and incubated at 32 ° C. At intervals of 24 hours each, an aliquot of 0.5 ml of medium was removed from each cavity and frozen instantaneously, and 0.5 ml of fresh medium was added to the cultures with p-trypsin. The virus in the aliquots was titrated at 32 ° C on LLC-MK2 cell monolayers using fluid casing as described previously (Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference). , and the end point of the titration was determined by hemadsorption and the titres were expressed as logioTCIDso / ml.

Replication of recombinant chimeric PIV3-PIV2 at various temperatures The viruses were serially diluted in IX L15 supplemented with 2 mM glutamine and 0.5 μg ml of p-trypsin. The diluted viruses were used to infect LLC-MK2 monolayers in 96-well plates. The infected plates were incubated at various temperatures for 7 days as described (Skiadopoulos et al., Vaccine 18: 503-510, 1999, incorporated herein by reference). The virus titers were determined as above.

Replication of immunogenicity and protective efficacy of recombinant chimeric PIV3-PIV2 viruses in the respiratory tract of hamsters Golden Syrian hamsters in groups of six were inoculated intranasally with 105"3TCID50 of the recombinant or biologically derived viruses.Four days after inoculation, the Hamsters were sacrificed and their nasal lungs and turbinates were harvested and prepared for virus quantification (Skiadopoulos et al., Vaccine 18: 503-510, 1999, incorporated herein by reference) .The titles were expressed as the mean logioTCIDso / gram of tissue for each group of six hamsters.The hamsters in groups of 12 were intransally infected with 105"3TCID5o of the viruses on day 0, and six hamsters of each group were inoculated four weeks later with 106TCID50 of PIV1 or 106TCID5o of PIV2. The hamsters were sacrificed 4 days after inoculation and their nasal lungs and turbinates were harvested. Titers of the inoculated virus were determined as described above (Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference). Virus titers were expressed as the mean logioTCID5o / gram of tissue for each group of six hamsters. Serum samples were collected three days prior to inoculation and on day 28, and antibody titers for haemagglutination inhibition antibody (HAI) against PIV1, PIV2 and PIV3 were determined as described previously (van Wyke Coelingh et al., Virology 143: 569-582,1985, incorporated herein by reference). The titles were expressed as the reciprocal mean log2.

Replication, immunogenicity and protective efficacy of the recombinant chimeric PIV3-PIV2 viruses in African green monkeys (A6M) The AGM in groups of 4 were infected intranasally and intratracheally with 105 TCID5o of the virus at each site on day 0. The specimens extracted by brush nasal / throat (NT) and tracheal washings were collected for 12 and 5 days, respectively, as described previously (van Wyke Coelingh et al., Virology 143: 569-582, 1985). On day 29, the immunized AGM were inoculated intranasally and intratrically with 105 TCID5o of PIV2 / V94 at each site. The specimens extracted by brush in nt and tracheal washings were collected during 10 and 5 days, respectively. Serum samples were collected before immunization, after immunization and after inoculation on days -3, 28 and 60, respectively. The titers of the virus in the specimens extracted by NT brush and in tracheal washings were determined as described above (Tao et al., J. Virol. 72: 2955-2961, 1998). The titles are expressed as logi0TCID5o / nil. Serum neutralizing antibody titers against PIV1 and PIV2 were determined as described above (van Wyke Coelingh et al., Virology 143: 569-582, 1985), and titers were expressed as the reciprocal mean log2.

Replication and immunogenicity of recombinant chimeric PIV3-PIV2 viruses in chimpanzees Chimpanzees in groups of 4 were infected intranasally and intratracheally with 105 TCID50 of PIV2 / V94 or PIV3r-2TM on day 0 as described previously (Whitehead et al., J. Virol. 72: 4467-4471, 1998, incorporated herein by reference). The specimens extracted by NT brush were collected daily for 12 days and obtained tracheal washings on days 2, 4, 6, 8 and 10. The virus titers in the specimens were determined as described previously (Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference). Peak virus titers were expressed as the mean logioTCID50 / nil. Serum samples were collected before immunization and after immunization on days -3 and 28, respectively. The titers of serum neutralizing antibody against PIV1 and PIV2 were determined as described above (van Wyke Coelingh et al., Virology 143: 569-582, 1985, incorporated herein by reference), and titers were expressed as the reciprocal mean log2.

The viable recombinant chimeric viruses were not recovered from the PIV3-PIV2 chimeric cDNA encoding the complete PIV2 F and HN proteins The construction of the PIV3-PIV2 chimeric cDNA, in which the F and HN ORFs of the PIV3 wild type JS were replaced by those of PIV2 / V94, described above and summarized in Figure 17. The final plasmid construct, pFLC.PIV32hc (Figure 17), which encodes a chimeric antigenic RNA of PIV3-PN2 of 15492 nt, which is conform to the rule of six. The HEp-2 cell monolayers were transfected with pFLC.PIV32hc together with the three support plasmids pTM (N), pTM (PnoC) and pTM (L) using LipofectACE, and the cells were simultaneously infected with MVA-T7 as described above (Tao et al., J. Virol. 72: 2955-2961, 1998, incorporated herein by reference). The virus was not recovered from the various initial transfections using pFLC.PN32hc, while the chimeric viruses were recovered from all transfections using the pFLC .2G + control plasmid. he. Consistent with these results is the possibility that a mutation occurred outside the 4 kb BspEI-Spel segment of pFLC.PIV32hc that prevented the recovery of PIV3r-2 virus from cells transfected with this cDNA clone. To examine this possibility, the BspEI-Spel fragments of p38'APIV31hc and p38'APIV32hc were exchanged. The regenerated p38'APIV31hc and p381 APIV32hc were identical to those of Figure 17 with the exception that the Spel-Sphl fragments containing the LIV3 gene sequences were exchanged. The BspEI-Sphl fragments of these two regenerated cDNAs were introduced into the BspEI-Sphl window of a full-length clone PIV3, p3 / 7- (131) 2G +, into five separate ligations separately to provide 10 clones of pFLC.2G +. hc and pFLC.PIV32hc (2 clones selected from each ligation) respectively. (Note that the PIV3 sequence outside the BspEI-Sphl window of p3 / 7 (131) 2G +, pFLC.2G + .hc and pFLC.PIV32hc are identical.) In this way, this could have replaced any sequence of the PIV3 structure. which could have acquired a spurious mutation with the known sequence to be functional. In addition, the functionality of the structure was reevaluated in parallel. None of the 10 cDNA clones pFLC.PIV32hc provided a viable virus, although each of the 10 cDNA clones pFLC.2G + .hc provided a viable virus. The virus was not recovered from pFLC.PIV32hc despite the step of harvesting tansfection in a manner similar to that successfully used to recover the recombinant C-separated from highly defective PIV3 (Durbin et al., Virology 261: 319-30, 1999, incorporated herein by reference). Because each of the components used to generate the pFLC. PIV32hc was used to successfully generate other recombinant viruses except for the cytoplasmic tail domains of F and HN, it is quite unlikely that errors in cDNA count for failure to provide recombinant virus in this case. Instead, the favored interpretation is that full length PIV2 F and HN glycoproteins are not compatible with one or more of the PIV3 proteins necessary for virus development.

Recovery of the chimeric viruses from the chimeric PIV3-PIV2 cDNAs coding for the chimeric PIV3-PIV2 F and HN proteins Using two different strategies, novel chimeric PIV3-PIV2 antigenomic cDNAs were constructed, in which the ectodomain or the ectodomain and the transmembrane domain of the F and HN glycoproteins of PIV3 were replaced by their PIV2 counterparts. The construction of the four length cDNAs, complete, namely pFLC. PIV32T, pFLC.PIV32TMcp45, pFLC. PIV32CT and pFLC. PIV32CTcp45, described above and summarized in Figures 18 19 and 20. The PIV3-2 inserts in the final plasmids pFLC.PIV32 ™ and pFLC. PIV32CT, in which the F and HN genes encoded for chimeric glycoproteins, were 15,498 nt and 15474 nt in length, respectively and conformed to the six rule (Calain et al., J. Virol. 67: 4822-30, 1993; Durbin et al., Virology 234: 74-83, 1997, each incorporated herein by reference). The authenticity of these four constructions was confirmed by sequencing the BspEI-Sphl region and by restriction analysis. The recombinant chimeric viruses were recovered from full-length clones pFLC. PIV32TM, pFLC. PIV32CT, pFLC. PIV32TMcp45 or pFLC.PIV32CTcp45 and were designated, PIV3r-2TM, PIV3r-2CT, PIV3r-2TMcp45 and PIV3r-2CTcp45, respectively. These viruses were biologically cloned by 3 consecutive terminal dilutions on Vero cells and then amplified three times on Vero cells.

Genetic characterization of recombinant chimeric PIV3-PIV2 viruses Biologically cloned chimeric PIV3-PIV2 viruses, PIV3r-2TM, PIV3r-2CT, PIV3r-2TMcp45 and PIV3r-2CTcp45 were propagated in LLC-MK2 cells and then concentrated. The viral RNAs extracted from the sedimented viruses were used in the RT-PCR amplification of specific gene segments using primer pairs specific for PIV2 or PIV3 (21, 22 or 23, 24 in Table 22). The restriction enzymatic digestion patterns of the RT-PCR products amplified with PIV2-specific primer pairs from PIV3r-2TM, PIV3r-2C, PIV3r-2TMcp45 and PIV3r-2CTcp45, were each distinct from those derived from PIV2 / V94 and their patterns, using EcoRI, Mfel, Ncol or PpuMI, were those expected from the designated cDNA. The nucleotide sequences for the 8 different PIV3-PIV2 junctions in the F and HN genes of PIV3r-2TM and PIV3r-2CT are given in Figure 20. Also, the cp45 tags present and PIV3r-2TMcp45 and PIV3r-2CTcp45, except those in the 3 'region and the start of the NP gene were verified with RT-PCR and restriction enzymatic digestion as described previously (Skiádopoulos et al., J Virol. 73: 1374-81, 1999, incorporated herein by reference). reference). These results confirmed the chimeric nature of the recovered PIV3-PIV2 viruses as well as the presence of the introduced cp45 mutations.

Recombinant chimeric viruses of PIV3-PIV2 are efficiently replicated in LLC-MK2 cells in vitro. The kinetics and magnitude of in vitro replication of the PIV3-PIV2 recombinant chimeric viruses were assessed by multiclick replication in LLC-MK2 cells (Figure 21). The cultures of LLC-MK2 cell monolayer in six-well plates were infected in triplicate with PIV3r-2TM, PIV3r-2CT, PIV3r-2TMcp45 or PIV3r-2CTcp45 at an MOI of 0.01 in the presence of p-trypsin (0.5 ug / ml) . The samples were removed from the culture supernatant at 24 hour intervals for 6 days. Each of the recombinant chimeric viruses, except PIV3r-2CTcp45 (clone 2A1), replicated at the same rate and at a similar level as their PIV2 / V94 precursor virus, indicating that the PIV3-PIV2 chimerization of F and HN proteins does not they altered the rates of development of the recombinant chimeric viruses and all reached a titer of 107 TCIDS0 / ml or higher. Only the PIV3r-2CTcp45 developed slightly faster in each of the two experiments and reached its peak titer before PIV2 / V94. This pattern of accelerated development was probably a result of an unidentified mutation in this clone because a sister clone failed to exhibit this pattern of development. Clone 2A1 of PIV3r-2CTcp45 was used in the studies described below.

The level of temperature sensitivity of the chimeric viruses PIV3r-2 and its cp45 derivatives The level of sensitivity to the replication temperature of the recombinant chimeric viruses PIV3-PIV2 was tested to determine whether the viruses PlV3r-2TM and PIV3r-2CT exhibit a ts phenotype and to determine whether the acquisition of the 12 cp45 mutations by these viruses specified a level of temperature sensitivity characteristic of cp45 derivatives carrying these 12 cp45 mutations of PIV3 (Skiadopoulos et al., J. Viro !. 73: 1374 -81,1999, incorporated herein by reference). The level of sensitivity to virus temperature was determined in MLC-2 cell monolayers as described above. (Skiadopoulos et al., Vaccine 18: 503-510,1999, incorporated herein by reference) (Table 26). The titer of PIV3r-2TM and PIV3r-2CT was completely constant at tolerant temperature (32 ° C) and the various restrictive temperatures tested indicated that these recombinants were ts +. In contrast, its derivatives cp45, PIV3r-2TMcp45 and PIV3r-2CTcp45, were ts and the level of sensitivity to temperature was similar to that of PIV3r-lcp45, the chimeric PIV3-PIV1 virus carrying the F and HN glycoproteins of PIV1 complete and the same set of 12 cp45 mutations. In this way, the in vitro properties of the irus PIV3r-2TM and PIV3r-2CT and its derivative cp45 are similar for those of PIV3r-l and PlV3r-lcp45, respectively, suggesting that the living properties of the PIV3r- viruses 2 and PIV3r-l could also be similar although surprisingly this was not the case.

Table 26. Replication of PIV3r-2CT and PIV3r-2TM was not temperature sensitive in LLC-MK2 cells, whereas the inclusion of cp45 mutations confers the temperature-sensitive phenotype cp45 Virus Title at 32 ° C Change in the title (logio) at various temperatures in (logio TCID50) compared with that at 32 ° superior, 35oc 36 ° 37 ° 38 ° 39 ° 40 ° PIV3r / JS 7.9 0.3b 0.1 0.1 (0.3) b (0.4) 0.4 PIV3cp45e 7.8 .0.5 0.3 1.3 3.4d 6.8 6.9 PIVl / Wash64e 8.5 1.5 1.1 1.4 0.6 0.5 0.9 PIV3r-l 8.0 0.8 0.5 0.6 0.9 1.1 2.6 PIV3r-lcp45 8.0 0.5 0.4 3.4d 4.8 6.6 7.5 PIV2 / V9412e 7.8 0.3 (0.1) 0.0 (0.4) (0.4) 0.0 PIV3r-2CT 6.9 0.3 0.3 0.6 (0.1) 0.6 0.4 PIV3r-2TM 8.3 0.3 (0.1) 0.3 0.6 1.0 2. ld PIV3r-2CTcp45 8.0 0.8 (0.4) 2.0d 4.3 7.5 > 7.6 PIV3r-2TMcp45 8.0 0.3 0.6 2.4d 5.4 7.5 > 7.6 The presented data are the means of two experiments. Numbers that are not in parentheses represent a decrease in the title; the numbers in parentheses represent an increase in the title. The data at 35 ° were from one experiment only. The values that are underlined represent the lowest temperature at which there was a 100-fold reduction in virus titre as compared to the titre at a tolerant temperature (32 ° C). This restrictive temperature is termed as the deactivation temperature. Biologically derived viruses.

PIV3r-2TM and PIV3r-2CT are attenuated, immunogenic and quite protective in hamsters, and the introduction of cp45 mutations results in fairly attenuated and less protective viruses. The hamsters in groups of six were inoculated intranasally with 105 3 TCID50 of PIV3r-2TM, PIV3r-2CT, PIV3r-2TMcp45, PIV3r-2CTcp45 or control virus. It was previously observed that the PIV3r-1 virus was replicated in the upper and lower respiratory tract of hamsters similar to that of PIV3 and PIV1 precursor (Skiadopoulos et al., Vaccine 18: 503-510, 1999; Tao et al., J. Virol. 72: 2955-2961, 1998, each incorporated herein by reference). The PIV2 virus replicates efficiently in hamsters, although the PIV3r-2T and PIV3r-2CT viruses each replicate to a lower titer of 50 to 100-fold than their PIV2 and PIV3 precursors in the upper respiratory tract and to a lower titer of 320 to 2000 times in the lower respiratory tract (Table 27). This indicates that chimeric PIV3-PIV2 F and HN glycoproteins specify an unexpected attenuation phenotype in hamsters. PIV3r-2T cp45 and PIV3r-2CTcp45, the derivatives that carry the cp45 mutations, were 50 to 100 times more attenuated than their respective PIV3r-2 with only barely detectable replication in the nasal turbinates and none in the lungs. These PIV3r-2cp45 viruses were clearly more attenuated than PIV3r-lcp45, which exhibits an additional 50-fold reduction in replication in the nasal turbinates. In this way, the attenuating effects of the F and HN glycoprotein chimerization and that specified by the cp45 mutations were additive.

Table 27. The PIV3r-2T and PIV3r-2CT viruses, in contrast to PIV3r-l, were attenuated in the respiratory tract of hamsters and the importation of the cp45 mutations resulted in an additional attenuation. - Virus titers in the indicated tissue (log10TCID50 / g ± S.E.) [Duncan group] Virus3 NT Lung reduction Title title reduction log10 log10 PIV3r / JS 5.9 0.1 [AB] 0 6.5 ± 0.1 [A] 0 PIV3rcp 5 4.5 ± 0.2. { C] 1.4C 1.8 ± 0.2 [E] 4.7C PIVl / Wash64d 5.7 O.lfB] - 5.5 ± O.lfB] - PIV3r-l 6.4 ± 0.2 [A] 0 6.6 ± 0.2 [A] 0 PIV3r-lCp45 3.1 ± 0.1 [D] 3.3C 1.2 ± 0.0 [F] 5.4C PIV2 / V94d 6.2 ± 0.2 [A] 0 6.4 ± 0.2 [A] 0 PIV3r-2CT 4.5 ± 0.4 [C] 1.7C 3.1 ± 0.1 [D] 3.3C PIV3r-2TM 3.9 ± 0.3 [C] 2.3C 3.9 ± 0.4 [C] 2.5C PIV3r-2CTcp45 1.4 ± 0.1 [E] 4.8C 1.5 ± 0.2 [E] 4.9C PIV3r-2TMcp45 1.6 ± 0.2 [E] 4.6C 1.4 ± 0.1 [E] 5.0C a Hamsters in a group of six were inoculated intranasally with 105'3 TCID50 of the virus indicated on day 0. b The hamsters were sacrificed and their tissue samples were collected on day 4. The virus titer in the hamster tissues was determined and the results are expressed as logioTCIDso / g ± standard error (SE). NT = nasal turbinates. c The logio reduction values were derived by comparison: PIV3rcp45 against PIV3r / JS; PIV3r-lcp45 against PIV3r-l; each of the PIV3-PIV2 chimeras against PIV2 / V94. d Biologically derived viruses. e Grouping as analyzed by the Duncan multiple test. To determine the immunogenicity and protective efficacy of the PIV3-PIV2 chimeric viruses / hamsters in groups of twelve were immunized with 105 · 3 TCID50 of PIV3r-2T, PIV3r-2CT, PIV3r-2TMcp45, PIV3r-2CTcp45 or control virus on the day 0. Six of the hamsters in each group were inoculated with 106 TCID50 of PIV1 on day 29 and the remaining half was inoculated with PIV2 on day 32. The hamsters were sacrificed 4 days after inoculation and the lungs and nasal turbinates were harvested. Serum samples were collected on day -3 and day 28 and their HAI antibody titer was determined against PIV1, PIV2 and PIV3. As shown in Table 28, despite their attenuated development of hamsters, immunization with PIV3r-2TM or PIV3r-2CT, each produced a level of serum HAI antibody against PIV2 which was comparable to that induced by infection with PIV2. / V94 wild type. Immunization of the hamsters with PIV3r-2TM and PIV3r-2CT resulted in a complete restriction in the replication of the PIV2 inoculated viruses. PIV3r-2TMcp45 and PIV3r-2CTcp45 failed to produce a detectable serum antibody response, and immunization of the hamsters with either of these two viruses resulted only in a 10 to 100 fold reduction in replication of the inoculated virus PIV2 in the lower respiratory tract (Table 28).

Table 28. PIV3r-2CT and PIV3r-2T viruses were quite protective in hamsters against inoculation with wild-type PIV2, although they were not against PIV1. indicated on day 0. b Serum samples were collected two days before immunization and 28 days after immunization. They were tested for HAI antibody titer against all three PIV and antibody titers were presented as the reciprocal mean log2 1 standard error (SE). c Six hamsters from each group were inoculated intranasally with 106 TCID50 of PIV1 (on day 29) or PIV2 (on day 32). The tissues of hamsters were harvested 4 days after inoculation and the virus titer in the indicated tissues was expressed as logio TCID50 / g and SE.

PIV3r-2TM and PIV3r-2CT are attenuated, immunogenic and quite protective in the AGM, whereas the introduction of the cp45 mutations resulted in the virus being rather attenuated and poorly protective. Certain recombinant PIV3 and RSV viruses can exhibit different levels of attenuation in rodents and primates (Skiadopoulos et al., Vaccine 1 ^ 8: 503-510, 1999, Skiadopoulos et al., J. Virol. 72: 1374-81, 1999a Skiadopoulos et al., Virology 272-34, 2000; Whitehead et al., J. Virol. 73; 9773-9780, 1999, each incorporated herein by reference), indicating that the attenuation may be specific in some species . Therefore, PIV3r-2 viruses were evaluated for their level of replication and immunogenicity in the AGM. The AGM in groups of four were intranasally and intratracheally administered 105 TCID50 per site of PIV3r-2TM, PIV3r-2CT, PIV3r-2TMcp45, piv3r-2CTcp 5, PIV2 / V94 or PIV3r-l on day 0. The virus in the specimens collected by NT brush (collected from day 1 to 12) and tracheal washings (collected on day 2, 4, 5, 8 and 10) were titrated as described previously (van Wyke Coelingh et al., Virology 143: 569-582 , 1985, incorporated herein by reference). As shown in Table 29, PIV3r-2TM and PIV3r-2CT were clearly attenuated in the respiratory tract of the AGM as indicated by a peak titer of the virus that spreads in both the upper and lower respiratory tracts. so does the PIV2 / V94 precursor virus. PIV3r-2TMcp45 and PIV3r-2CTcp45, the derivatives that carry the cp45 mutations were detected at very low levels, if any, in specimens extracted by NT swab and tracheal wash, which suggests that the attenuating effects of glycoprotein chimerization F and HN and that specified by the cp45 mutations were additives for the AGM as well as for the hamsters. To determine whether immunization of the AGM with the chimeric viruses PIV3-PIV2 was protective against inoculation with PIV2, the AGM previously infected with a PIV3r-2 virus were inoculated with 105 TCID50 of PIV2 on day 28 (Table 29). The virus present in specimens extracted by NT brush (collected from day 29 to 38) and tracheal wash fluids (collected on day 30, 32, 34, 36 and 38) was titrated as described previously (Durbin et al. , Virology 261: 319-30, 1999, incorporated herein by reference). As shown in Table 29, immunization with PIV3r-2 ™ and PIV3r-2CT induced a high level of restriction of replication of the inoculated virus PIV2 / V94. In contrast, immunization of AGM with PlV3r-2TMcp45 and PIV3r-2CTcp45 failed to restrict replication of the inoculated PIV2 / V94 virus and these animals developed very low levels of serum neutralization antibodies prior to inoculation for PIV2. The complete restriction of replication of the PIV2 / V94 inoculated virus in the AGMs immunized against PIV3r-2CT is associated with a level 2.5 higher than the serum antibody previously inoculated for PIV2 than that of the AGM immunized with PIV3r-2TM which provides incomplete protection.

Table 29. PIV3r-2CT or PIV3r-2TM viruses were attenuated for replication in the respiratory tract of African green monkeys, still induce resistance to inoculation with wild-type PIV2 Virus Mediated titre tb antibody Title of Immunizing peak titer3 immunizing virus serum neutralization of the virus at the indicated site against the virus inoculated PIV2 / V94 at logio TCID5o / ml ± SE) indicated (mean the indicated site (logio reciprocal log2 ± SE) TCID5o / ml ± SE) 1í NT 1 TL PIV1 PIV2 1 NT 1 1 TL PIV3r-l 2 .6 ± 0 .5 3 .2 ± 0. 1 6 .3 ± 0. 4 3.1 + 0.3 3 .6 ± 0. 2 3 .310. 7 PIV2 / V94 2 .8 ± 0 .7 5 .0 ± 0. 3 3 .8+ 0. 0 7.1 ± 0.7 < 0.2 = 0.2 PIV3r-2CT 1 .5 ± 0 .4 0 .5 ± 0. 2 2 .910. 1 7.2 ± 0.1 = 0.2 = 0.2 PIV3r-2TM 1 .4 ± 0 .1 1 .6 + 0. 7 4 .1 ± 0. 1 5.9 + 0.2 1 .6 ± 0. 6 1 .3 ± 0. 9 PIV3r-2CTcp45 1 .0 ± 0 .2 = 0.2 4 .1 ± 0. 1 . 5.310.0 3 .3 ± 0. 4 3 .5 ± 0. 3 PIV3r-2TMcp45 0 .6 ± 0 .3 < 0.2 3 .4 ± 0. 2 4.6 + 0.6 3 .0 + 0. 5 4 .1 ± 0. 2 The African green monkeys in groups of 4 were inoculated with 105 TCID50 of the indicated virus intranasally and intratracheally on day 0. The combined samples extracted by nasal lavage and by throat swab (NT) were collected on days 1 to 12. Tracheal washout (TL) samples were collected on days 2, 4, 6, 8 and 10. Virus titers were determined in LLC-MK2 monolayers and were expressed as logio TCID5o / ml ± standard error (SE). The serum samples collected on day 28 were analyzed for their neutralizing antibody titers against PIV1 and PIV2. The titles were expressed as the reciprocal mean log2 ± SE. NT specimens were collected on days 29 to 38. TL specimens were collected on days 30, 32, 34, 36 and 38.

PIV3r-2TM is attenuated in its replication in the respiratory tract of chimpanzees Chimpanzees in groups of 4 were inoculated intranasally and intratracheally with 105 TCID50 or 105 TCID50 of PIV3r-2TM or PIV2 / V94 on day 0. Specimen samples were collected by NT swab (day 1 to 12) and tracheal wash (days 2, 4, 6, 8 and 10). The virus titer was determined as described previously (Durbin et al., Virology 261: 319-30, 1999, incorporated herein by reference) and the results are expressed as log10 TCID50 / ml. As shown in Table 30, the PIV3r-2TM had a lower peak titer than that of the PIV2 / V94 wild type precursor and spreads for a significantly shorter duration than the PIV2 / 94, indicating that PIV3r-2TM is attenuated in chimpanzees The wild-type PIV2 / 94 virus replicates at low levels in chimpanzees compared to hamsters and AFGs, while the PIV3r-2T virus is attenuated in each of these model hosts.

Table 30. PIV3r-2TM is attenuated in the respiratory tract of chimpanzees and still produces an important serum immune response for PIV2 Media Virus of the titb peak of the Mean of the Virus antibody titer scattered at the site of serum neutralizing virus days against indicated ( logio TCIDso / ml ± that spreads the indicated virus (mean SE) in the reciprocal tract log2 ± SE) upper respiratory (days NT TL ± SE) PRE POST PIV2 / V94 2.9 ± 0.6 1.2 ± 0.5 8.8 ± 1.1a = 2.8 ± 0.0 6.2 ± 0.5 PIV3r-2TM 2.0 ± 0.3 = 0.5 ± 0.0 2.5 ± 0.7a 3.3 ± 0.2 4.3 ± 0.4 a Chimpanzees in groups of four were inoculated intranasally and intratracheally with 105 TCID50 of the indicated virus. b Specimens extracted by nose / throat swab (NT) and tracheal lavage (TL) were collected for 12 and 10 days, respectively, and virus titers were determined. Peak titers were expressed as logio TCID50 standard error (SE). c Serum samples collected before 3 days before and 28 days after virus inoculation were analyzed for their neutralizing antibody titer against the indicated virus. The titles were expressed as the reciprocal mean log2 ± SE. d Significant difference in the duration of recreation, p = 0.005. Student's T test.

As noted above, the protective antigens of PIVs are their HN and F glycoproteins. This form, in exemplary embodiments of the invention, viruses for PIV candidate vaccine attenuated in vivo for use in infants and young children include HPIV3 viruses. -1 and chimeric HPIV3-2 carrying the full length PIV1 and PIV2 glycoproteins, respectively, in a PIV3 background or antigenome genome. In the latter case, the chimeric HN and F ORFs instead of the full-length PIV2 ORFs are used to construct the full-length cDNA. The recovered viruses, designated PIV3r-2CT in which the PIV2 ectodomain and the transmembrane domain fuse for the cytoplasmid domain PIV3 and PIV3r-2TM in which the PIV2 ectodomain is fused to the PIV3 transmembranes and the cytoplasmic tail domain, possessed similar in vitro phenotypes and in vivo. In particular, the recombinant chimeric viruses PIV3r-2 exhibited a host classification phenotype, that is, they replicated efficiently in vitro although they were restricted in in vivo repliciation. This attenuation in vivo occurs in the absence of any aggregated mutations of cp45. That is, an unexpected host classification effect that is quite desirable for vaccine purposes, in part because the phenotype is specified by point mutations, which may be referred to as wild type. At the same time, unregistered in vitro development is quite advantageous for efficient vaccine production. Although PIV3r-2CT and PIV3r-2TM replicate efficiently in vitro, they are quite attenuated in the upper and lower respiratory tracts of hamsters and African green monkeys (AGM), indicating that the chimerization of PIV2 HN and F proteins and PIV3 themselves specify an in vivo attenuation phenotype. In spite of this attenuation, they are quite immunogenic and protective against inoculation with the wild type PIV2 virus in both species. PIV3r-2CT and PIV3r-2TM were further modified by introducing the 12 PIV3 cp45 mutations located outside the HN and F coding sequences to derive PIV3r-2CTcp45 and PIV3r-2TMcp45 which efficiently replicated in vitro but were even further attenuated in hamsters and AGM which indicates that it is the specific attenuation by the glycoprotein chimerization and by the cp45 mutations was additive. The development of antigenic chimeric viruses possessing virus-protecting antigens and attenuating mutations of another virus has been reported by others for influenza viruses (Belshe et al., N. Engl. J. Med. 338: 1405-1, 1998; Murphy et al., Infectious Diseases in Clinical Practice 2: 174-181, 1993) and for rotaviruses (Perez-Schael et al., N. Engl. J. Med. 337: 1181-7, 1997). Attenuated antigenic vaccines are more easily generated by these viruses that have segmented genomes, because reclassification of the genomic segment occquite frequently during coinfection. Candidates for influenza virus vaccine attenuated annually are antigenically updated by replacing the HA and NA genes of the attenuated donor virus with those of a new epidemic or pandemic virus. Recombinant DNA technology is also actively used to construct live antigen attenuated chimeric virus vaccines for flaviviruses and for paramyxoviruses. For flavivirus a candidate has been produced for live attenuated virus vaccine for Japanese encephalitis virus (JEV) by replacing the premembrane (prM) and envelope (E) regions of attenuated yellow fever virus (YFV) with those of an attenuated strain of JEV (Guirakhoo et al., Virology 257: 363-72, 1999). The candidate for JEV-YFV antigenic chimeric recombinant vaccine is attenuated and immunogenic in vivo (Guirakhoo et al., Virology 257: 363-72, 1999). The structural and non-structural proteins of this chimeric virus come from a live attenuated vaccine virus. Chimeric antigenic vaccines have also been produced between a flavivirus that carries a naturally attenuated function (Langat virus) and a dengue 4 virus carrying a wild type mosquito and the resulting recombinant was found to be significantly more attenuated for mice than its virus carrying a precr function (Pletnev et al., Proc Nati Acad Sci USA 95: 1746-51, 1998), although this chimeric virus was quite restricted in replication in Vero cells in vitro. This is an example of an attenuating effect of the partial incompatibility between the evolutionary divergent structural proteins specified by the Langar virus and the nonstructural proteins of the dengue virus. A third strategy is being ped for the production of a vaccine for the dengue virus quatrivalent in which a structure of dengue 4 containing an attenuating deletion mutation in the 3 'non-coding region is used to construct antigenic chimeric viruses containing protective antigens of dengue 1, 2 or 3 viruses (Bray et al., Proc Nati Acad Sci USA 88: 10342-6, 1991, J. Virol 70: 3930-7, 1996). Chimeric antigenic viruses have been produced for RNA viruses in the negative sense, single chain. For example, candidates for antigenic chimeric PIV1 vaccine can be constructed according to the methods set forth herein by substituting the proteins HN and F of parainfluenza virus type 1 for those of PIV3 in a candidate for attenuated PIV3 vaccine and this recombinant is attenuated and the protector against the inoculation of PIV1 of experimental animals. Similarly, candidates for exemplary antigenic chimeric respiratory syncytial virus (RSV) vaccine can be produced in which one or more of the RSV protective F and G antigens or antigenic determinants thereof, of subgroup B virus is they substitute for those in an attenuated RSV A subgroup virus that provide candidates for attenuated RSV subgroup B vaccine. (See also, International Publication No. WO 7/06270; Collins et al., Proc. Nati, Acad. Sci. USA 92: 11563-11567 (1995); United States Patent Application No. 08 / 892,403; filed on July 15, 1997 (corresponding to published International Application No. WO 98/02530 and United States Provisional Requests of Priority Nos. 60 / 047,634, filed May 23, 1997, 60 / 046,141, filed on May 9, 1997 and 60 / 021,773, filed July 15, 1996); United States Patent Application No. 09/291, 894, filed by Collins et al on April 13, 1999; U.S. Provisional Patent Application Serial No. 60 / 129,006, filed April 13, 1999; U.S. Provisional Patent Application Serial No. 60 / 143,132, filed by Bucholz et al. on July 9, 1999 and Whiteheád et al., J. Virol. 73: 9773-9780, 1999, each incorporated herein by reference). When the glycoprotein exchanges between the PIV1 and PIV3 viruses and between the RSV subgroup A and RSV subgroup B viruses were performed in a wild type virus background, the antigenic chimeric viruses replicated to wild type virus levels in vitro and in vivo. These findings indicate that a high level of compatibility exists between the recipient and donor viruses and that only very little, if any, is achieved as a result of the chimerization process. These findings with the glycoprotein A and B exchanges of PIV1 and PIV3 and RSV contrast impressively in various ways with those between PIV2 and PIV3 described herein. In the present exposition, the viable recombinant virus in which the full-length PIV2 HN or F protein is used to replace those of PIV3 was not recovered in this case, evidently what can be compensated for incidental mutations introduced during the construction of the CDNA while this was successfully achieved for the glycoprotein exchange of PIV1-PIV3. This suggests that the HN or F glycoprotein of PIV2 is poorly compatible with one or more of the PIV3 proteins encoded in the cDNA. Two PIV2-PIV3 chimeric viruses were obtained when the chimeric HN and F ORFs instead of the full-length PIV2 ORF were used to construct the full-length cDNA. One of these chimeric viruses contained the chimeric HN and F glycoproteins in which the PIV2 ectodomain was fused for the PIV3 and cytoplasmic transmembrane tail region and the other contained the chimeric HN and F glycoproteins in which the PIV2 ectodomain and the region are fused. transmembrane to the cytoplasmic tail region PIV3. Both PIV3r-2 recombinants possessed similar but not identical phenotypes in vitro and in vivo. In this way, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required to successfully recover the chimeric viruses of PIV2-PIV3. In previous studies aimed at analyzing the structural function of the protein, chimeric HN or F proteins have been constructed and expressed in vitro and used to map several functional domains of the proteins (Bousse et al., Virology 204: 506 -14, 1994, Deng et al., Arch. Virol. Suppl. 13: 115-30, 1997; Deng, et al., Virology 253: 43-54, 1999; Deng et al., Virology 209: 457-69. , 1995, Mébatsion et al., J. Vlrol., 69: 1444-1451, 1995, Takimoto et al., J. Virol. 72: 9747-54, 1998, Tanabayashi et al., J. Virol. 70: 6112- 6118, 1996, Tsurudome et al., J. Gen. Virol. 79: 279-89, 1998, Tsurudome et al., Virology 213: 190-203, 1995, Yao et al., J. Virol. 69: 7045- 53, 1995). In one report, a chimeric glycoprotein consisting of a cytoplasmic tail F of measles virus fused to the transmembrane and the ectodoniin of the G protein of the vesicular stomatitis virus was inserted into an infectious clone of the measles virus instead of the glycoproteins F and HN of the measles virus (Spielhofer et al., J. Virol. 72: 2150-9, 1998). A chimeric virus was obtained that was competent in replication, although quite restricted in in vitro replication as indicated in the delayed development and by low virus yields which indicates a high degree of in vitro attenuation. This finding is in marked contrast to the phenotype exhibited by the recombinant PIV of the invention which expresses chimeric glycoproteins, for example a PIV2-PIV3 chimera, which replicates efficiently in vitro. Efficient replication of the PIV3r-2 viruses and other chimeric PIVs of the invention in vitro is an important property for a candidate for attenuated vaccine, live, which is necessary for the production of large-scale vaccine. In contrast to PIV3r-2CT and PIV3r-2TM, PIV3r-l was not attenuated in vivo. In this way, the chimerization of the HN and F proteins of PIV2 and PIV3 itself resulted in an attenuation for replication in vivo, a novel finding for RNA viruses in the negative sense, single-stranded. The mechanism for this host classification restriction of in vivo replication is known. Importantly, infection with these candidates for PIV3r-2CT and PIV3r-2TM vaccine induced a high level of resistance to inoculation with PIV2 indicating that the antigenic structure of the chimeric glycoproteins was quite or completely intact. In this way, the function PIV3r-2CT and PIV3r-2TM as the viruses for candidate vaccine PIV2 attenuated in viruses, exhibited a desirable balance between attenuation and immunogenicity in both the AGM and hamsters. The attenuating effects of the PIV3-PIV2 chimerization of the F and HN glycoproteins are additive with those specified by the cp45 mutations. The PIV3r-2 recombinants containing the cp45 mutations were quite attenuated in vivo and provided incomplete protection in hamsters against inoculation with PIV2 and a small protection in the AGM. This is in contrast to the finding with PIV3r-lcp45 that was successfully attenuated in vivo and protected animals against inoculation with PIV1. The combination of the additive attenuating effects independent of the chimerization of the PIV3-PIV2 glycoproteins and the 12 cp45 mutations seemed too attenuating in vivo. Clearly, if candidates for PIV3r-2CT and PIV3r-2TM vaccine were found to be insufficiently attenuated in humans, attenuating mutations of cp45 must be added incrementally rather than a set of 12 to achieve a desired balance between the attenuation and immunogenicity necessary for an attenuated PIV2 vaccine in vivo for use in humans. The findings presented herein identify a novel means to attenuate a paramyxovirus and provide the basis for the evaluation of these candidates for PIV2 attenuated chimeric live PIV3-PIV2 vaccine in humans. Importantly, the PIV3r-2CT or PIV3r-2TM viruses can also be used as vectors for other PIV antigens or for other viral protective antigens, for example, the HA protein of the measles virus or the immunogenic portions thereof. Briefly summarizing the description and the above examples, recombinant chimeric PIV constructed as vectors carrying heterologous viral genes or genomic segments have been produced and characterized using a cDNA based virus recovery system. The recombinant viruses produced from the cDNA replicate independently and can be propagated in the same way as if they were biologically derived viruses. In preferred embodiments, candidates for recombinant chimeric human PIV (HPIV) vaccine carry one or more antigenic determinants of an HPIV, preferably an antecedent that is attenuated by one or more nucleotide modifications. Preferably, the chimeric PIVs of the invention also express one or more protective antigens of another pathogen, for example, a microbial pathogen. In these cases, HPIV acts as an attenuated virus vector and is used for the dual purpose of inducing a protective immune response against one or more of the HPIV as well as against the pathogens from which the foreign protective antigens are derived. As mentioned above, the main protective antigens of PIVs are their HN and F glycoproteins. The main protective antigens of other enveloped viruses, for example viruses that infect the respiratory tract of humans, which can be expressed by the HPIV vector from one or more extra transcriptional units, they are also referred to as gene units, are their binding proteins, for example, the G protein of RSV, the protein HA of measles virus, the protein HN of mumps virus or its fusion proteins (F), for example , the RSV F protein, the measles virus or the mumps virus. It is also possible to express the protective antigens of the non-enveloped viruses such as for example the Ll protein of the human papillomaviruses which could form the virus-like particles in the infected hosts (Roden et al., J. Virol. 70: 5875 -83, 1996). In accordance with these teachings, a large series of protective antigens and their constituent antigenic determinants can be integrated from various pathogens within the chimeric PIV of the invention to generate novel, effective immune responses. The present invention overcomes the difficulties inherent in the above proposals for the development of the vaccine based on the vector and provides unique opportunities for the immunization of infants during the first year of life against a variety of human pathogens. Previous studies in the development of attenuated PIV vaccines in vivo indicating, unexpectedly, rIVIV and its attenuated and chimeric derivatives have properties that make them uniquely suitable among non-segmented negative-strand RNA viruses as vectors for expressing foreign proteins as vaccines against a variety of human pathogens. The expert may not have predicted that human PIVs, which tend to develop substantially less well than non-segmented negative chain viruses and that have usually been poorly represented with respect to molecular studies, might prove to have characteristics that are quite favorable as vectors. It is also surprising that the intranasal administration route of these vaccines has proven to be a very efficient means to stimulate an important local and systemic immune response both against the vector and against the expressed heterologous antigen. In addition, this route provides additional advantages for immunization against heterologous pathogens that infect the respiratory tract or elsewhere. These properties of the PIV vectors were described hereinbefore using examples of the PIV3r vectors that carry (i) an antigen from the main neutralization of the measles virus expressed as a separate gene in wild type and attenuated background or (ii) antigens from primary neutralization of HPIV1 in place of PIV3 neutralizing antigens that additionally express a primary neutralization antigen of HPIV2. These rIVr vectors are constructed using wild-type and attenuated background. In addition, the description of the present demonstrates the ability to easily modify the attenuation level of the PIV vector structure. According to one of these methods, the variation in the length of agreement of the genomic inserts in a chimeric PIV of the invention allows adjustment of the attenuation phenotype, which is only evident in the wild-type derivatives using very large inserts. The present invention provides six main advantages over previous attempts to immunize the small infant against the measles virus or other microbial pathogens. First, the recombinant vector of PIV in which the antigen or protective antigens of measles virus or other microbial pathogens is inserted is an attenuated PIVr carrying one or more attenuating genetic elements that are known to attenuate viruses for the respiratory tract of the very young human infant (Karron et al., Pediatr. Infect. Dis. J. 15: 650-654, 1996; Karron et al., J. Infect. Dis. 171: 1107-1114, 1995a; Karron et al., J. Infect. Dis. 172: 1445-1450, 1995b). This extensive history of previous clinical evaluation and practice greatly facilitates the evaluation of the derivatives of these recombinants that carry the above protective antigens in the very young human infant. The second advantage is that the structure of rIVr is carried in measles HA or another protective antigen of another human pathogen will induce a dual protective immune response against (1) the PIV, for which there is a mandatory independent need for a vaccine as indicated above, and (2) the heterologous virus or another microbial pathogen whose protective antigen is expressed by the vector. This contrasts with the recombinant HA of the measles virus VSV described above which will induce immunity only for a human pathogen, i.e., the measles virus and in which the immune responses for the vector itself are either irrelevant or potentially disadvantageous. The coding sequences of the above genes inserted into several members of the order of Mononegavirales virus have remained intact in the genomes of most of the recombinant viruses after multiple cycles of replication in tissue culture cells, indicating that the members of these groups of viruses are excellent candidates for use as vectors (Bukreyev et al., J. Virol. 70: 6634-41, 1996; Schnell et al., Proc. Nati, Acad. Sci. USA 93: 11359-65, 1996a; Singh et al., J. Gen. Virol. 80: 101-6; Yu et al., Genes Cells 2: 457-66, 1997). Another advantage provided by the invention is the use of a human pathogen structure, for which a vaccine is needed, will favor the introduction of this vector of attenuated virus in vivo in an immunization scheme in early childhood already overloaded. In addition, immunization through the mucosal surface of the respiratory tract offers several advantages. A PIV3 attenuated in vivo was shown to replicate in the respiratory tract of the rhesus monkeys and induce an immunoprotective response against itself in the presence of large amounts of PIV3 antibodies acquired maternally. The ability of the two candidate PIV3 vaccines to infect and efficiently replicate in the upper respiratory tract the very young human infant who possesses acquired antibodies via the mother was also demonstrated (Karron et al., Pediatr. Infect. Dis. J. 15: 650-654, 1996, Karron et al., J. Infect. Dis. 171: 1107-1114, 1995a, Karron et al., J. Infect. Dis. 172: 1445-1450, 1995b). This is in contrast to the currently authorized measles virus vaccine which is poorly infective when administered to the upper respiratory tract of humans and which is quite sensitive to neutralization when administered parenterally to young children (Black et al., New Eng. J. Med. 263: 165-169, 1960; Kok et al., Trans. R. Soc. Trpp. Med. Hyg. 77: 171-6, 1983; Simasathien et al., Vaccine l_5: 329- 34, 1997). Replication of the HPIV vector in the respiratory tract will stimulate the production of both IgA mucosal and sistamic immunity for the HPIV vector and for the expressed anterior antigen. At the time of subsequent natural exposure to the wild-type virus, for example measles virus, the existence of the immunity induced by the local and systemic vaccine should serve to restrict its replication and its oral entry, ie, respiratory tract, thus as well as in systemic replication sites. Yet another advantage of the invention is that chimeric HPIVs carrying the heterologous sequence replicate efficiently in vitro demonstrating the ease of large-scale production of the vaccine. This is a contrast to the replication of some RNA viruses in the negative sense of individual chain that can be inhibited in vitro by the insertion of a foreign gene. { Bukreyev et al., J. Virol. 70: 6634-41, 1996). As well, the presence of three HPIV antigenic serotypes, each of which causes significant disease in humans and therefore can simultaneously serve as a vector and vaccine, presents a unique opportunity to sequentially immunize the infant with antigenically distinct HPIV variants each one reports the same strange protein. In this way, the sequential immunization will allow the development of an immunoprimary response to the foreign protein that can be increased during subsequent infections with the antigenically distinct HPIV that also carries the same or different foreign proteins, that is, the protective antigen of the virus. measles or another microbial pathogen. It is also likely that readministration of homologous HPIV vectors will also increase the antigen response of both HPIV and foreign due to the ability to induce multiple reinfections in humans is an unusual but characteristic attribute of HPIV (Collins et al., In. "Fields Virology", BN Fields, DM Knipe, PM Howley, RM Chanock, JL Melnick, TP Monath, B. Roizman, and S. Straus, Eds., Vol. 1, pp. 1205-1243. Lippincott-Raven Publishers, Philadelphia, 1996). Yet another advantage is that the introduction of the gene unit into the PIV vector has several unexpected, though quite desirable, defects for the production of the attenuated viruses. First, the insertion of the gene units expressing, for example, the HA of the measles viruses or the HN of PIV2 can specify a host classification phenotype on the PIV vector that has not been previously recognized, i.e. the resulting PIV vector replicates efficiently in vitro although it is restricted in in vivo replication in both upper and lower respiratory tracts. These findings identify the insertion of a gene unit expressing a viral protective antigen as an attenuating factor for the PIV vector, a desirable property in the live attenuated virus vaccines of the invention. The PIV vector system has unique advantages with respect to all other members of the viruses in the negative sense of individual chain of the Mononegavirales order. First, most of the various mononegaviruses that have been used as vectors are not derived from human pathogens (e.g., murine HPIV1 (Sendai virus) (Sakai et al., FEBS Lett 456: 221-6, 1999). , vesicular stomatitis virus (VSV) which is a bovine pathogen (Roberts et al., J. Virol. 72: 4704-11, 1998) and canine PIV2 (SV5) He et al., Virology 237: 249-60, 1997 )). For these non-human viruses, a small or only weekly immunity could be conferred against any human virus by the antigens present in the vector structure. Thus, a non-human virus vector expressing a supernumerary gene for a human pathogen could induce resistance only against that individual human pathogen. In addition, the use of viruses such as the virus VSV, SV5, rabies or Sendai as a vector could expose vaccines to viruses that they probably would not otherwise meet during life. The infection with, and the immune responses against, these non-human viruses may be of marginal benefit and could have safety concerns, because there is little experience of infection with these viruses in humans. An important and specific advantage of the PIV vector system is that its preferred intranasal administration route, which mimics natural infection, induces both mucosal and systemic immunity and reduces the neutralizing and immunosuppressive effects of maternally derived serum IgG that is present in infants . While these disadvantages are theoretically possible to use RSV as a vector, for example, it has been found that RSV replication is significantly inhibited by inserts greater than about 500 base pairs (Bukreyev et al., Proc. Nati Acad. Sci. USA 96: 2367-72, 1999). By contrast, as described herein, the HPIV3 can be easily adapted to various large gene inserts. The finding that RSV recoiminant is unsuitable for carrying these large inserts, while recombinant PIVs are quite adequate, represents unexpected results. It could be proposed that some other viral vector could be administered intranasally to obtain similar benefits as those shown for the PIV vectors, although this has not been successful to date. For example, the MVA strain of vaccinia virus expressing the HPIV3 protective antigens was evaluated as an attenuated intranasal vaccine in vivo against HPIV3. Although this vector appears to be highly efficient in the expression system in cell culture, it was inexplicably inefficient in inducing a resistance in the primate upper respiratory tract (Durbin et al., Vaccine 16: 1324-30, 1998) and inexplicably it was inefficient to induce a protective response in the presence of passive serum antibodies (Durbin et al., J. Infect. Dis. 179: 1345-51, 1999). In contrast, it has been found that candidates for PIV3 and RSV vaccine are protective in the upper and lower respiratory tract of non-human primates, even in the presence of passive serum antibodies (Cro et al., Vaccine 13: 847-855, 1995).; Durbin et al., J. Infect. Dis. 17_9_: 1345-51, 1999). The use of PIV3 in particular as a vector still offers additional advantages. For example, conditions have been established to obtain high titers of PIV3 in microcarrier culture that are 10 to 1000 times greater than what can be achieved with viruses such as for example RSV and measles virus. Also, the RSV infection capacity is unstable, which complicates propagation, transport, storage and handling. These problems will be obvious through the development of an RSV vaccine vectorized with PIV. Importantly, the two versions of PIV3 have undergone extensive clinical evaluation, candidate vaccines administered intranasally, through BP1V3 and the attenuated HPIV3 cp45 strain. It was found that each one is safe, immunogenic and phenotypically stable in children and infants. No other vector designed as a candidate was evaluated in children and infants and in particular no other available vector has been evaluated for intranasal administration in this age group. Another advantage of the PIV vector system is that, by using HPIV3, as a model, several attenuating mutations have been identified that can be introduced into the vector structure individually and in combination to obtain the desired degree of attenuation. For example, specific mutations conferring the cp45 attenuation phenotype of HPIV3 have been directly identified by sequence analysis and introduction into wild-type recombinant virus. Additional attenuating mutations were developed by attenuating point mutations "importation" of the Sendai virus and RSV. In some cases, it was possible to introduce certain point mutations in the recombinant virus using two nucleotide changes instead of one, which stabilizes the mutation against wild-type reversion. It showed that the withdrawal of the expression of the ORF of C, D and V attenuates the virus. In addition, HPIV3 chimeric viruses and bovine (B) PIV3 were developed to utilize the natural host classification restriction of BPIV3 in primates as a means of attenuation. It was also found that certain sequence combinations were attenuating, such as the replacement of the HN and F ectodomains of HPIV3 with their HPIV2 counterparts. Thus, there is a large menu of PIV attenuating mutations that can be used to attenuate the vector structure as desired. Thus, one aspect of the invention disclosed herein relates to a method for using the recombinant PIVs selected as vectors to express one or multiple protective antigens of a heterologous pathogen as supernumerary genes. The heterologous pathogens described herein include heterologous PIV, measles virus and RSV. In the above examples, the HPIV3r was designed as a vector to express up to three separate supernumerary gene inserts each expressing a different viral protective antigen. In addition, the HPIV3r easily adapts to the total aggregate insert length of at least 50% that of the wild-type genome. Constructs were made with different vector structures of different PIV, namely: wild type HPIV3; an attenuated version of HPIV3 in which the ORF of N was replaced by that of BPIV3; the HPIV3-1 chimeric virus, in which the HPV3 HN and F ORFs were replaced by their HPIV1 counterparts; a version of HPIV3-1 that was attenuated by the presence of three independent attenuating cp45 point mutations in the L gene; and a version of BPIV3 in which the HN and F genes were replaced by their HPIV3 counterparts. These vectors carrying one or more supernumerary genes were replicated efficiently in vitro, demonstrating feasibility for their commercial development and they replicated and induced important immune responses in vivo against both the vector and the inserts. In this way it is possible to construct a virus based on individual recombinant PIV that is able to induce an immune response against at least four human pathogens, namely the PIV vector itself and the pathogens represented by the supernumerary genes. A second aspect of the invention is to use the superior characteristics of the PIV as a vaccine and as a vector to produce a vaccine against RSV. RSV is a pathogen that develops less well than PIV, is unstable and tends to induce immune responses that are poorly protective for reasons they do not fully understand. The development of an attenuated RSV vaccine has been on the road for more than 35 years, indicating the difficulty in achieving an adequate balance between immunogenicity and attenuation for this human pathogen. Thus, there are compelling reasons to develop a live attenuated RSV vaccine that is not based on infectious RSV. The main protective F and G antigens against RSV were expressed as supernumerary genes of the PIV vector, in this case BPIV3, which makes obvious the need to produce an attenuated vaccine in vivo based on the infectious RSV. A third aspect of the invention described herein has been to develop PIV-based vectors that carry the antigenic determinants of different PIV serotypes. Because there is essentially no cross-protection between serotypes, this makes it possible to develop a method for sequential immunizations with a common PIV vector in which protective antigenic determinants are changed. In this way, a simple attenuated PIV vector structure such as for example derived from HPIV3r, which carries supernumerary genes as desired, can be used for an initial immunization. A subsequent immunization, which preferably follows the first of 4 a 6 or more weeks, can be achieved by using a version of the same PIV vector in which the vector glycoprotein genes have been replaced with those of a heterologous PIV serotype, such as by example in HPIV3r-1. This vector may contain the same supernumerary genes, which could then provide a "boost" against the supernumerary antigens, or may contain a different set. Because the second immunization is performed with a version of the vector containing the glycoproteins of a heterologous PIV serotype, there is some interference by the specific immunity of the vector induced by the initial immunization. Alternatively, the second immunization can be performed with a PIV vector in which all the vector genes are of different serotypes, such as for example HPIV1 or HPIV2. However, the advantage of using a common set of internal genes, such as for example in vectors PIV3r and PIV3r-l which are based on HPIV3, is that a simple set of attenuating mutation can be employed in each construct, and there is no need to separately develop attenuated strains of each PIV serotype. Importantly, sequential immunization follows an anti-viral strategy: in each immunization, the vector itself induces immunity against the important human pathogen in each supernumerary insertion gene, induces immunity against an additional pathogen. Although the above invention has been described in detail by way of example for purposes of clarity and understanding, it will be apparent to the skilled person that certain changes and modifications may be practiced within the scope of the appended claims which are presented by way of illustration and not as a limitation. In this context, various publications and other references have been cited within the previous discussion for economy of description. Each of these references is incorporated herein by reference in its entirety for all purposes.

DEPOSIT OF BIOLOGICAL MATERIAL The following materials have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, in accordance with the terms of the Budapest Treaty.

Virus Access No. Deposit date p3 / 7 (131) 2G (ATCC 97989) April 18, 1997 p3 / 7 (131) (ATCC 97990) April 18, 1997 p218 (131) (ATCC 97991) April 18, 1997 cp45 JS of HPIV3 ( ATCC PTA-2419) August 24, 2000 LIST OF SEQUENCES < 110 > THE GOVERNMENT OF THE UNITED STATES OF AMERICA, such as Murp and, Brian R. Collins, Peter L. Schmidt, Alexander C. Durbin, Anna P. Skiadopoulos, Mario H. Tao, Tao < 120 > USE OF RECOMBINANT PARINFLUENZA VIRUSES (PIVS) AS VECTORS FOR THE PROTECTION AGAINST INFECTION AND DISEASE CAUSED BY PIV AND OTHER HUMAN PATHOGENS < 130 > 15280-404100PC < 140 > < 141 > < 150 > 60/170, 195 < 151 > 1999-12-10 < 150 > 09 / 458,813 < 151 > 1999-12-10 < 150 > 09/459, 062 < 151 > 1999-12-10 < 160 > 62 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 41 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Flanking sequence of the measles HA gene insert for N-P and P-M < 400 > 1 cttaagaata tacaaataag aaaaacttag gattaaagag cg 42 < 210 > 2 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Flanking sequence of the measles HA gene insert for N-P and P-M < 400 > 2 gatccaacaa agaaacgaca ccgaacaaac cttaag 36 < 210 > 3 < 211 > 101 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Flanking sequence of the HA gene insert of measles for splicing HN-L < 400 > 3 aggcctaaaa gggaaatata aaaaacttag gagtaaagtt acgcaatcca actctactca 60 tataattgag gaaggaccca atagacaaat ccaaattcga g 101 < 210 > 4 < 211 > 79 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Flanking sequence of the measles HA gene insert for splicing HN-L < 400 > 4 tcataattaa ccataatatg catcaatcta tctataatac aagtatatga taagtaatca 60 gcaatcagac aataggcct 79 < 210 > 5 < 211 > 64 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Cloning site for insertion GU < 400 > 5 aggaaaaggg aaatataaaa aacttaggag taaagttacg cgtgttaact tcgaagagct 60 ccct 64 < 210 > 6 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Cloning site for insertion NCR < 400 > 6 aggaaaaggg aacgcgtgtt aacttcgaag agctccct 38 < 210 > 7 < 211 > 63 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Cloning site for the supernumerary gene insert between the P and M genes of HPIV3r < 400 > 7 ttaacaatat acaaataaga aaaacttagg attaaagagc catggcgtac gaagcttacg 60 cgt 63 < 210 > 8 < 211 > 12 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Gene end sequence (GE) of PIV3 < 400 > 8 aagtaagaaa aa 12 < 210 > 9 < 211 > 58 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Cloning site for inserts of the GV and F gene of RSV in B / H PIV3 < 400 > 9 aggattaaag aactttaccg aaaggtaagg ggaaagaaat cctaagagct tagcgatg 58 < 210 > 10 < 211 > 11 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Flanking sequence of the GV insert of RSV in B / H PIV3 < 400 > 10 gcttagcgat g 11 < 210 > 11 < 211 > 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Flanking sequence of the GV and F gene inserts of RSV in B / H PIV3 < 400 > 11 aagotagogo ttagc 15 < 210 > 12 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Flanking sequence of the F gene insert of RSV in B / H PIV3 < 400 > 12 gcttagoaaa aagctagcae aatg 24 < 210 > 13 < 211 > 83 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Direct primer for PCR of the measles HA gene insert for the N-P and P-M < 400 > 13 ttaatcttaa gaatatacaa ataagaaaaa cttaggatta aagagcgatg tcaccacaac 60 gagaccggat aaatgccttc tac 83 < 210 > 14 < 211 > 67 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse primer for PCR of the measles HA gene insert for the N-P and P- < 400 > 14 attattgctt aaggtttgtt cggtgtcgtt tctttgttgg atcctatctg cgattggttc 60 catcttc 67 < 210 > 15 < 211 > 55 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Direct primer for PCR of the measles HA gene insert for the HN-L < 400 > 15 gacaataggc ctaaaaggga aatataaaaa acttaggagt aaagttacgc aatcc 55 < 210 > 16 < 211 > 68 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse / direct primer for PCR of the measles HA gene insert for the HN-L < 400 > 16 gtagaacgcg tttatccggt ctcgttgtgg tgacatctcg aatttggatt tgtctattgg 60 gtccttcc 68 < 210 > 17 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse primer for PCR of the measles HA gene insert for the HN-L < 400 > 17 ooatgtaatt gaatcoccca acactagc 28 < 210 > 18 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Direct / reverse primer for PCR of the measles HA gene insert for the HN-L < 400 > 18 cggataaaog cgttotaoaa agataaco 28 < 210 > 19 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: HPV2 HN Primer 5 '< 400 > 19 gggccatgga agattacagc aat 23 < 210 > 20 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HN Primer of HPIV2 in the 3 'direction < 400 > 20 caataagctt aaagcattag ttcoo 25 < 210 > 21 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: HPV2 HN Primer 5 '< 400 > 21 gcgatgggcc cgaggaagga cccaatagac to 31 < 210 > 22 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HPIV2 primer in the 3 'direction < 400 > 22 coogggtcct gatttcocga geaogctttg 30 < 210 > 23 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HPV1 HN Primer < 400 > 23 agtggctaat tgcattgcat ccacat 26 < 210 > 24 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: HPV1 HN Primer < 400 > 24 gccgtctgca tggtgaatag caat 24 < 210 > 25 < 211 > 13 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligomeric insert for compliance of the rule of six < 400 > 25 ogoggoaggc ctg 13 < 210 > 26 < 211 > 14 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligomeric insert for compliance of the six rule < 400 > 26 cgcggcgagg cctg 14 < 210 > 27 < 211 > 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligomeric insert for compliance of the rule of six < 400 > 27 cgogaggcct ocgcg 15 < 210 > 28 < 211 > 16 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligomeric insert for compliance of the rule of six < 400 > 28 egogccgcgg aggcct 16 < 210 > 29 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligomeric insert for compliance of the rule of six < 400 > 29 cgcgcccgcg gaggcct 17 < 210 > 30 < 211 > 42 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > . Description of the Artificial Sequence: Direct primer for the RSV gene A G insert < 400 > 30 aattcgctta gcgatgtaca aaagga ccaacgcaac go 42 < 210 > 31 < 211 > 92 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Reverse primer of the RSV gene A G insert < 400 > 31 aaaaagctaa gcgctagcct ttaatcctaa gtttttctta ctttttttac tactggcgtg 60 gtgtgttggg tggagatgaa ggttgtgatg gg 92 < 210 > 32 < 211 > 65 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Direct primer of the RSV gene A F insert < 400 > 32 aaaggcctgc ttagcaaaaa gctagcacaa tggagttgct aatcctcaaa gcaaatgcaa 60 ttacc 65 < 210 > 33 < 211 > 89 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Reverse primer of the RSV gene A G insert < 400 > 33 aaaagctaag cgctagcttc tttaatccta agtttttctt acttttatta gttactaaat 60 gcaatattat ttataccact cagttgatc 89 < 210 > 34 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutagenic direct primer for the modification of cDNA of HPIV3r-l < 400 > 34 cggcagtgac gcgtctccgc accggtgtat taagcogaag caaa 44 < 210 > 35 < 211 > 59 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Mutagenic Inverse Primer for the Modification of HPIV3r-l &ct; 400 > 35 cccgagcacg etttgctcct aagtttttta tatttcccgt acgtctattg tctgattgc 59 < 210 > 36 < 211 > 95 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Direct primer for the insertion of ORF F of HPIV2 in the genome of B / HPIV3r < 400 > 36 aaaatatagc ggccgcaagt aagaaaaact taggattaaa ggcggatgga tcacctgcat 60 ccaatgatag tatgcatttt tgttatgtac actgg 95 < 210 > 37 < 211 > 72 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse primer for the insertion of ORF F of HPIV2 in the genome of B / HPIV3r < 400 > 37 aaaatatagc ggccgctttt actaagatat cccatatatg tttccatgat tgttcttgga 60 aaagacggca gg 72 < 210 > 38 < 211 > 81 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Direct primer for the insertion of HF ORF of HPIV2 in the genome B / HPIV3r < 400 > 38 ggaaaggcgc gccaaagtaa gaaaaactta ggattaaagg cggatggaag attacagcaa 60 tctatctctt aaatcaattc c 81 < 210 > 39 < 21'1 > 54 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse primer for the insertion of O F HN of HPIV2 in the genome B / HPIV3r < 400 > 39 ggaaaggcgc gccaaaatta aagcattagt tcccttaaaa atggtattat ttgg 54 < 210 > 40 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV2 (sense) < 400 > 40 gtaccatgga tcacotgcat ccaat 25 < 210 > 41 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV2 (antisense) < 400 > 41 tgtggatcct aagatatccc atatatgttt c 31 < 210 > 42 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV2 F (sense) < 400 > 42 atgcatcacc tgoatccaat < 210 > 43 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, PIV2 (antisense) < 400 > 43 tagtgaataa agtgtcttgg ct 22 < 210 > 44 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, HN of PIV2 (sense) < 400 > 44 catgagataa ttcatcttga tgtt 24 < 210 > 45 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, HN of PIV2 (antisense) < 400 > 45 agcttaaago attagttccc ttaa 24 < 210 > 46 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV3 (sense) < 400 > 46 atcataatta ttttgataat gatoatta 28 < 210 > 47 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV3. { antisense) < 400 > 47 gttcagtgot tgttgtgtt 19 < 210 > 48 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, HN of PIV3 (sense / antisense) < 400 > 48 tcatáattaa ccataatatg catcaat 27 < 210 > 49 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, HN of PIV3 (sense) < 400 > 49 gatggaatta attagcaota tgat 24 < 210 > 50 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV2 (antisense) < 400 > 50 atgcatoacc tgcatccaat < 210 > 51 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV2 (sense) < 400 > 51 gatgatgtag gcaatcago 19 < 210 > 52 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, HN of PIV2 (sense) < 400 > 52 aetgacaoaa ttcttggc 18 < 210 > 53 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the PIV3-2 HN chimeric cDNAs of PIV2 (antisense) < 400 > 53 ttaaagoatt agttcoctta aaaatg 26 < 210 > 54 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV3 (sense) < 400 > 54 aagtattaca gaattcaaaa gag 23 < 210 > 55 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > '< 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, HN of PIV3 (antisense) < 400 > 55 ottattagtg agc tgttgc 20 < 210 > 56 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, F of PIV2 (sense) < 400 > 56 acogaagotg tagcaatagt 20 < 210 > 57 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, HN of PIV2 (antisense) < 400 > 57 gattcca ca cttaggtaaa t 21 < 210 > 58 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of the chimeric cDNAs of PIV3-2, M of PIV3 (sense) < 400 > 58 gatactatcc taatattatt ge 22 < 210 > 59 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer for the construction of PIV3-2.L chimeric cDNAs of PIV3 (antisense) < 400 > 59 gctaattttg atagcacatt 20 < 210 > 60 < 211 > 15492 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Sequence of pFLC. PIV32, 15492 bp in sense orientation < 400 > 60 accaaacaag agaagaaact tgtctgggaa tataaattta actttaaatt aacttaggat 60 taaagacatt gactagaagg tcaagaaaag ggaactctat aatttcaaaa atgtgagcc 120 tatttgatac atttaatgca cgtaggcaag aaaacataac aaaatcagcc ggtggagcta 180 tcattcctgg acagaaaaat actgtctcta tattcgccct tggaccgaca ataactgatg 240 aatgacatta ataatgagaa gctcttctat ttctatctca ttcactagat aatgagaaac 300 aacatgcaca aagggcaggg ttcttggtgt ctttattgtc aatggcttat gccaatccag 360 agctctacct aacaacaaat ggaagtaatg cagatgtcaa gtatgtcata tacatgattg 420 agaaagatct aaaacggcaa aagtatggag gatttgtggt taagacgaga gagatgatat 480 atgaaaagac aactgattgg atatttggaa gtgacctgga ttatgatcag gaaactatgt 540 tgcagaacgg caggaacaat tcaacaattg aagaccttgt ccacacattt gggtatccat 600 catgtttagg agctcttata atacagatct ggatagttct ggtcaaagct atcactagta 660 tctcagggtt aagaaaaggc tttttcaccc gattggaagc tttcagacaa gatggaacag 720 tgcaggcagg gctggtattg agcggtgaca gattgggtca cagtggatea atcatgcggt 780 ctcaacagag cttggtaact cttatggttg aaacattaat aacaatgra & t accagcagaa 840 atgacctcac aaccatagaa aagaatatac aaattgttgg caactacata agagatgcag 900 gtctcgcttc attcttcaat acaatcagat atggaattga gaccagaatg gcagctttga 960 tctcagacca ctctatccac gatatcaata gattaaaagc tttgatggaa ctgtatttat 1020 caaagggacc acgcgcteet ttcatctgta tcctcagaga tcctatacat ggtgagttcg 1080 caccaggcaa ctatcctgcc atatggagct atgcaatggg ggtggcagtt gtacaaaata 1140 gagccatgca acagtacgtg acgggaagat catatctaga cattgatatg ttccagctag 1200 gacaagcagt agcacgtgat gccgaagctc aaatgagctc aacactggaa gatgaacttg 1260 cgaatctaaa gagtgacaca gaaagcttga agagacatat aaggaacata aacagttcag 1320 agacatcttt ccacaaaccg acaggtggat cagccataga gatggcaata gatgaagagc 1380 cgaacataga cagaacaatt gcagatcaag aacaaaatgg agaacctcaa tcatccataa 1440 ctgggcagaa ttcaatatgc ggaaatagaa gcgatgatca gactgagcaa gctacagaat 1500 ctgaca atat caacaaaaca caagaccgaa tcagagacag actaaacaag agactcaacg 1560 acaaagcagt acaagaagaa caaccaccca ctaatcccac aaacagaaca aaccaggacg 1620 aaatagatga tctgtttaac gcatttggaa gcaactaatc gaatcaacat tttaatctaa 1680 aataagaaaa at.caat.aata acttaggatt aaagaatcct atcataccgg aatatagggt 1740 ggtaaattta gagtctgctt gaaactcaat caatagagag ttgatggaaa gcgatgctaa 1800 atcatggatt aaactatcaa cttgggaaga ggaatcaaga gataaatcaa ctaatatctc 1860 ctcggccctc aacatcattg aattcatact cagcaccgac ccccaagaag acttatcgga 1920 atcaacacaa aaacgacaca gaacccagca actcagtgcc accatctgtc aaccagaaat 1980 caaaccaaca gaaacaagtg agaaagatag tggatcaact gacaaaaata gacagtccgg 2040 gaatgtacaa gtcatcacac cagaagcaaa agatagaaat attgatcagg aaactgtaca 2100 gggagaagaa gagaggacct gcagctcaga tagtagagct gagaetgtgg tctctggagg 2160 a &tccccaga agcatcacag attctaaaaa tggaacccaa aacacggagg atattgatct 2220 caatgaaatt agaaagatgg ataaggactc tattgagggg aaaatgcgac aatctgcaaa 2280 tgttccaagc gagatatcag gaagtgatga catatttaca acagaacaaa gtagaaacag 2340 tgatca tgga agaagcctgg aatctatcag tacacctgat acaagatcaa taagtgttgt 2400 tactgctgca acaccagatg atgaagaaga aatactaatg aaaaatagta ggacaaagaa 2460 aagttcttca acacatcaag aagatgacaa aagaattaaa aaagggggaa aagggaaaga 2S20 ctggtttaag aaatcaaaag ataccgacaa ccagatacca acatcagact acagatccac 2580 atcaaaaggg cagaagaaaa tctcaaagac aacaaccacc aacaccgaca caaaggggca 2640 aacagaaata cagacagaat catcagaaac acaatcctca tcatggaatc tcatcatcga 2700 caacaacacc gaccggaacg aacagacaag cacaactcct ccaacaacaa cttccagatc 2760 aaagaatcga aacttataca tccgaacaaa aaacccaaga ctctgaatcc cacaaaagac 2820 aaatggaaag gaaaggaagg atacagaaga gagcaatcga tttacagaga gggcaattac 2880 tctattgcag aatcttggtg taattcaatc cacatcaaaa ctagatttat atcaagacaa 2940 tgtgtagcaa acgagttgta atgtactaaa caatgtagat actgcatcaa agatagattt 3000 cctggcagga ttagtcatag gggtttcaat ggacaacgac cacagataca acaaaattaa 3060 aaatgaaatg ctaaacctca aagcagatct aaagaaaatg gacgaatcac atagaagatt 3120 gatagaaaat caaagagaac aactgtcatt gatcacgtca ctaatttcaa atctcaaaat 3180 tatgactgag agaggaggaa agaaagacca aaatgaatcc aatgagagag tatccatgat 3240 caaaacaaaa ttgaaagaag aaaagatcaa gaagaccagg tttgacccac ttatggaggc 3300 gacaagaata acaaggcatt tacccgatct atatcgacat gcagga gata cactagagaa 3360 cgatgtacaa gttaaatcag agatattaag ttcatacaat gagtcaaatg caacaagact 3420 aaagtgagca aatacccaaa gtacaatgag atcactagtt gcágtcatea acaacagcaa 3480 tctctcacaa agcacaaaac aatcatacat aaaegaaetc aaacgttgca aaaatgatga 3540 agaagtatct gaattaatgg acatgttcaa tgaagatgtc aacaattgcc aatgatccaa 3600 acaccgaaca caaagaaacg aacagacaag aaacaacagt agatcaaaac ctgtcaacac 3660 acacaaaatc aagcagaatg aaacaacaga tatcaatcaa tatacaaata agaaaaactt 3720 aggattaaag aataaattaa tccttgtcca aaatgagtat aactaactct gcaatataca 3760 cattcccaga atcatcattc tctgaaaatg gtcatataga accattacca ctcaaagtca 3840 gaaagcagta atgaacagag ccccacatta gagttgccaa gatcggaaat ccaccaaaac 3900 acggatcccg gtatttagat gtcttcttac tcggcttctt cgagatggaa cgaatcaaag 3960 acaaatacgg gagtgtgaat gatctcgaca gtgacccgag ttacaaagtt tgtggctctg 4020 gatcattacc aatcggattg gctaagtaca ctgggaatga ccaggaattg ttacaagccg 4080 caaccaaact ggatatagaa cagtcaaagc gtgagaagaa gaaagagatg gttgtttaca 4140 tataaaacca cggtacaaaa gaactgtacc catggtccaa tagactaaga aaaggaatgc 4200 tgttcgatgc caacaaagtt gctcttgctc ctcaatgtct tccactagat aggagcataa 4260 aatttagagt aatcttcgtg aattgtacgg caattggatc aataaccttg ttcaaaattc 4320 ggcatcacta ctaagtcaat tctctaccca aatcaatctg acacaatatc caggtacaca 4380 taaaaacagg ggttcagact gattctaaag ggatagttca aattttggat gagaaaggcg 4440 aaaaatcact gaatttcatg gtccatctcg gattgatcaa aagaaaagta ggcagaatgt 4S00 actctgttga atactgtaaa cagaaaatcg agaaaatgag attgatattt tctttaggac 560 tagttggagg aatcagtctt catgtcaatg caactgggtc catatcaaaa acactagcaa 4620 gtcagctggt attcaaaaga gagatttgtt atcctttaat ggatctaaat ccgcatctca 4680 ctgggcttca atctagttat tcagtagaga ttacaagagt ggatgcaatt ttccaacctt 4740 ctttacctgg cgagttcaga tactatccta atattattgc aaaaggagtt gggaaaatca 4800 aacaatggaa ctagtaatct ctattttagt ccggacgtat ctattaagcc gaagcaaata 4860 aaggataatc aaaaacttag gacaaaagag gtcaataeca acaactatta gcagtcacac 4920 tcgcaagaat aagagagaag ggaccaaaaa agtcaaatag gagaaatcaa aacaaaaggt 4980 agaacaacaa acagaacacc aatcaaaaca tccaactcac tcaaaacaaa AATTCC aaaa 5040 gagaccggca acacaacaag cactgaacac catggatcac ctgcatccaa tgatagtatg 5100 catttttgtt atgtacactg gaattgtagg ttcagatgcc attgctggag atcaactcct 5160 caatgtaggg gtcattcaat caaagataag atcactcatg tactacactg atggtggcgc 5220 tagctttatt gttgtaaaat tactacccaa tcttccccca agcaatggaa catgcaacat 5280 gatgcacata caccagccca atgttaccct atttaagttg ctaacacccc tgattgagaa 5340 cctgagcaaa atttctgctg ttacagatac caaaccccgc cgagaacgat ttgcaggagt 5400 cgttattggg cttgctgcac taggagtagc tacagctgca caaataaccg cagctgtagc 5460 gccaatgcaa aatagtaaaa atgctgctgc gataaacaat cttgcatctt caattcaatc 5520 caccaacaag gcagtatccg atgtgataac tgcatcaaga acaattgcaa ccgcagttca 5580 agcgattcag gatcacatca atggagccat tgtcaacggg ataacatctg catcatgccg 5640 tgcccatgat gcactaattg ggtcaatatt aaatttgtat ctcactgagc ttactacaat 5700 atttcataat caaataacaa accctgcgct gacaccactt tccatccaag ctttaagaat 5760 cctcctcggt agcaccttgc caattgtcat tgaatccaaa ctcaacacaa aactcaacac 5820 agcagagctg ctcagtagcg gactgttaac tggtcaaata atttccattt ccccaatgta 5 880 catgcaaatg ctaattcaaa tcaatgttcc gacatttata atgcaacccg gtgcgaaggt 5940 attgctatct aattgatcta ctgcaaacca taaattacaa gaagtagttg tacaagttcc 6000 taatagaatt ctagaatatg caaatgaact acaaaactac ccagccaatg attgtttcgt 6060 gacaccaaac tctgtatttt gtagatacaa tgagggttcc ccgatccctg aatcacaata 6120 tcaatgctta agggggaatc ttaattcttg cacttttacc cctattatcg ggaactttct 6180 gcatttgcca caagcgattc atggtgtgct ctatgccaac tgcaaatctt tgctatgtaa 6240 gtgtgccgac cctccccatg ttgtgtctca agatgacaac gcataattga caaggcatca 6300 tattaagagg tgctctgaga cactttttca tgatgcttga tttaggatca catctacatt 6360 caatgctaca tacgtgacag acttctcaat gattaatgca aatattgtac atctaagtcc 6420 tctagacttg tcaaatcaaa tcaattcaat aaacaaatct cttaaaagtg ctgaggattg 6480 gattgcagat agcaacttct tcgctaatca agccagaaca gccaagacac tttattcact 6540 aagtgcaatc gcattaatac tatcagtgat tactttggtt gttgtgggat tgctgattgc 6600 aagctggttt ctacatcatc ctcaaatcca tcaattcaga gcactagctg ctacaacaat 6660 gttccacagg gagaatcctg ccgtcttttc caagaacaat catggaaaca tatatgggat 6720 atctt aggat ccctacagat cattagatat taaaattata aaaaacttag gagtaaagtt 6780 actctactca acgcaatcca tataattgag gaaggaccca atagacaaat ccaaatccat 6840 ggaagat ac agcaatctat ctcttaaatc aattcctaaa aggacatgta gaatcatttt 6900 ccgaactgcc acaattcttg gcatatgcac attaattgtg gtattcttca ctatgttcaa 6960 tgagataatt catcttgatg tttcctctgg tcttatgaat tctgatgagt cacagcaagg 7020 cattattcag cctatcatag aatcattaaa atcattgatt gctttggcca accagattct 7080 atataatgtt gcaatagtaa ttcctcttaa aattgacagt atcgaaactg taatactctc 140 tgctttaaaa gatatgcaca ccgggagtat gtccaatgcc aactgcacgc caggaaatct 7200 gcttctgcat gatgcagcat acatcaatgg aataaacaaa ttccttgtac ttgaatcata 7260 caatgggacg cctaaatatg gacctctcct aaatataccc agctttatcc cctcagcaac 7320 atctccccat gggtgcacta gaataccatc attttcactc atcaagaccc atcggtgtta 7380 cactcacaat gtaatgcttg gagattgtct tgatttcacg gcatctaacc agtatttatc 7440 aatggggata atacaacaat ctgctgcagg gtttccaatt ttcaggacta tgaaaaccat 7500 gatggaatca ttacctaagt atcgcaaaag ctgttcagtc actgctatac caggaggttg 7560 tgtcttgtat t gctatgtag ctacaaggtc tgaaaaagaa gattatgcca cgactgatct 7620 agctgaactg agacttgctt tctattatta taatg tacc tttattgaaa gagtcatatc 7680 tcttccaaat acaacagggc agtgggccac aatcaaccct gcagtcggaa gcgggatcta 7740 tcatctaggc tttatcttat ttcctgtata tggtggtctc ataaatggga ctacttctta 7800 caatgagcag tcctcacgct attttatccc aaaacatccc aacataactt gtgccggtaa 7860 caggctgcaa ctccagcaaa tagcacggag ttcctatgtc atccgttatc actcaaacag 7920 gttaattcag agtgctgttc ttatttgtcc attgtctgac atgcatacag aagagtgtaa 7980 tctagttatg tttaacaatt cccaagtcat gatgggtgca gaaggtaggc tctatgttat 8040 tggtaataat ttgtattatt atcaacgcag ttcctcttgg tggtctgcat cgctctttta 8100 caggatcaat acagattttt ctaaaggaat tcctccgatc attgaggctc aatgggtacc 8160 gtcctatcaa gttcctcgtc ctggagtcat gccatgcaat gcaacaagtt tttgccctgc 8220 taattgcatc acaggggtgt acgcagatgt gtggccgctt aatgatccag aactcatgtc 8280 ctgaacccca acgtaatgct actatcgatt tgctggagcc tttctcaaaa atgagtccaa 8340 ccgaactaat cccacattct acactgcatc ggctaactcc ctcttaaata ctaccggatt 8400 aatcaca caacaacacc aag cagcatatac atcttcaacc tgctttaaaa acactggaac 8460 tattgtttaa ccaaaaaatt aatgggctca taataattga gggagttcca tctcttttag 8520 aataatacca tttttaaggg aactaatgct ttaagcttaa ttaaccataa tatgcatcaa 8580 atacaagtat tctatctata atgataagta agacaataga atctgcaatc caaaagggaa 8640 cttaggagca atataaaaaa aagcgtgctc gggaaatgga aacaatggca cactgaatct 8700 ctgtatctga catactctat cctgagtgtc accttaactc tcctatcgtt aaaggtaaaa 8760 tagcacaatt acacactatt atgagtctac ctcagcctta tgatatggat gacgactcaa 8820 tactagttat cactagacag aaaataaaac ttaataaatt ggataaaaga caacgatcta 8880 aaaattaata ttagaagatt ttaactgaaa aagtgaatga tacacattta cttaggaaaa 8940 agaaatgtca tcagatatcc aaagaaatgt tcaaattata attaacagta tatacctggt 9000 attattactt aagtgactga aaagcagata gaacatatag tcaaatgact gatggattaa 9060 gagatctatg gattaatgtg ctatcaaaat tagcctcaaa aaatgatgga agcaattatg 9120 atcttaatga agaaattaat aatatatcga aagttcacac aacctacaaa tcagataaat 9180 attcaaaaca ggtataatcc tggtttacta tcaagtatga tatgagaaga ttacaaaaag 9240 gatcactttt ctcgaaatga aa tgttggga aggattataa cttgttagaa gaccagaaga 9300 atttcttatt gatacatcca gaattggttt tgatattaga taaacaaaac tataatggtt 9360 atctaattac tcctgaatta gtattgatgt attgtgacgt agtcgaaggc cgatggaata 9420 taagtgcatg tgctaagtta gatccaaaat tacaatctat gtatcagaaa ggtaata & Cc 9480 gatagataaa tgtgggaagt ttgtttccaa ttatgggaga aaagacattt gatgtgatat 9540 accacttgca cgttattaga ttcaaactca ttatccttaa tgatcctgtt aaacaaetaa 9600 gaggagcttt tttaaatcat gtgttatccg agatggaatt aatatttgaa tctagagaat 9660 atttctgagt cgattaagga gtagattaca ttgataaaat tttagatata tttaataagt 9720 tgaaatagca ctacaataga gagattttct ctttttttag aacatttggg catcctccat 9780 tattgcagca tagaagctag gaaaatatat gaaaaggtta gtatattgga aaacaattaa 9840 aatttgacac tattaataaa tgtcatgcta tcttctgtac aataataatt aacggatata 9900 gagagaggca tggtggacag tggectcctg tgacat acc tgatcatgca cacgaattca 9960 ttacggttca tcataaatgc aactctgcga tatcatatga aaatgctgtt gattattacc 10020 agagctttat aggaataaaa ttcaataaat tcatagagcc tcagttagat gaggatttga 10080 gaaagataaa caatttatat gcattatctc caaaaaaatc aaattgggac acagtttatc 10140 ctgcatctaa tttactgtac cgtactaacg catccaacga atcacgaaga ttagttgaag 10200 tatttatagc agatagtaaa tttgatcctc atcagatatt ggattatgta gaatctgggg 10260 tgatccagaa actggttaga tttaatattt cttatagtct taaagaaaaa gagatcaa ac 10320 aggaaggtag actctttgca aaaatgacat acaaaatgag agctacacaa gttttatcag 10380 agaccctact tgcaaataac ataggaaaat tctttcaaga aaatgggatg gtgaagggag 10440 agattgaatt ttaacaacca acttaagaga tatcaatatc aggagttcca cggtataatg 10500 taattctaaa aagtgtacaa agecatacag atgacettaa aacctacaat aaaataagta 10560 atottaattt gtcttctaat cagaaatcaa attcaagtca agaaatttga acggatatct 10620 acaatgatgg atacgagact gtgagctgtt tcctaacaac agatcteaaa aaatactgtc 10680 ttaattggag atatgaatca acagctctat ttggagaaac ttgcaaceaa atatttggat 10740 taaataaatt gtttaattgg ttacacectc gtcttgaagg aagtacaatc tatgtaggtg 10800 atccttactg tcctccatca gataaagaac atatatcatt agaggatcac cctgattctg 10860 tcataaccca gtttttacgt agagggggta tagaaggatt ttgtcaaaaa ttatggacac 10920 tcatatctat aagtgcaata catctagcag ctgttagaat aggcgtgagg gtgactgcaa 10980 tggttcaagg agacaatcaa gctatagctg taaccacaag agtacccaac aattatgact 11040 gaaggagata acagagttaa gtttataaag atgtagtgag attttttgat tcattaagag 11100 tgatctaggt aagtgatgga catgaactta aattaaatga aacgattata agtagcaaga 11160 tagcaaaaga tgttcatata atctattatg atgggagaat tcttcctcaa gctctaaasg 11220 cattatctag atgtgtcttc tggtcagaga cagtaataga cgaaacaaga tcagcatctt 11280 caaatttggc aacatcattt gcaaaagcaa ttgagaatgg ttattcacct gttctaggat 11340 atgcatgctc aatttttaag aatattcaac aactatatat tgcccttggg atgaatatca 11400 atccaactat aacacagaat atcagagatc agtattttag gaatccaaat tggatgcaat 11460 atgcctcttt aatacctgct agtgttgggg gattcaatta catggccatg tcaagatgtt 11520 ttgtaaggaa tattggtgat ccatcagttg ccgcattggc tgatattaaa agatttatta 11580 aggcgaatct attagaccga agtgttcttt ataggattat gaatcaagaa ccaggtgagt 11640 catctttttt ggactgggct tcagatccat attcatgcaa tctcaaaata tttaccacaa 11700 taaccaccat gataaaaaat ataacagcaa ggaatgtatt acaagattca ccaaatccat 11760 tattatctgg attattcaca aatacaatga tgaagaatta tagaagaaga gctgagttcc 11820 tgatggacag gaaggtaatt ctccctagag ttgcacatga tattctagat aattctctca 11880 aaatgccata caggaattag gctggaatgt tagatacgac aaaatcacta attcgggttg 11940 aat gcataaatag aggaggactg acatatagtt tgttgaggaa cagtaat tacgatctag 12000 tacaatatga aacactaagt aggactttgc gactaattgt aagtgataaa atcaagtatg 12060 aagatatgtg ttcggtagac cttgccatag cattgcgaca aaagatgtgg attcatttat 12120 caggaggaag gatgataagt ggacttgaaa cgcctgaccc attagaatta ctatctgggg 12180 tagtaataac aggatcagaa cattgtaaaa tatgttattc ttcagatggc acaaaeccat 12240 ataettggat gtatttaccc ggtaatatea aaataggatc agcagaaaca ggtatatcgt 12300 cattaagagt tccttatttt ggatcagtca ctgatgaaag atctgaagca caattaggat 12360 tcttagtaaa atatcaagaa cctgcaaaag ccgcaataag aatagcaatg atatatacat 12420 gggcatttgg taatgatgag atatcttgga tggaagectc acagatagca caaacacgtg 12480 caaattttac actagatagt ctcaaaattt taacaccggt agctacatca acaaatttat 12540 cacacagatt aaaggatact gcaactcaga tgaaattctc cagtacatca ttgatcagag 12600 tcagcagatt eataacaatg tccaatgata acatgtctat caaagaagct aatgaaacca 12660 aagatactaa tcttatttat caacaaataa tgttaacagg attaagtgtt ttcgaatatt 12720 tatttagatt aaaagaaacc acaggacaca accctatagt tatgcatctg cacatagaag 12780 atgagtgttg tattaaagaa agttttaatg ATGAA catat taatccagag tctacattag 12840 aattaattcg atatcctgaa agtaatgaat ttatttatga taaagaccca ctcaaagatg 12900 tggacttatc aaaacttatg gttattaaag accattctta cacaattgat atgaattatt 12960 gggatgatac tgacatcata catgcaattt caatatgtac tgcaattaca atagcagata 13020 attagatcga ctatgtcaca gataatttaa aagagataat agttattgca aatgatgatg 13080 atattaatag cttaatcact gaatttttga ctcttgacat acttgtattt ctcaagacat 13140 ttggtggatt attagtaaat caatttgcat acactcttta tagtctaaaa atagaaggta 13200 gggatctcat ttgggattat ataatgagaa cactgagaga tacttcccat tcaatattaa 13260 taatgcatta aagtattatc tctcatccta aagtattcaa gaggttctgg gattgtggag 13320 ttttaaaccc tatttatggt cctaatactg ctagtcaaga ccagataaaa cttgccctat 13380 atattcacta ctatatgtga gatctattta tgagagaatg gttgaatggt gtatcacttg 13440 aaatatacat ttgtgacagc gatatggaag ttgcaaatga taggaaacaa gcctttattt 13500 ctagacacct ttcatttgtt tgttgtttag cagaaattgc atctttcgga cctaacctgt 13560 taaacttaac atacttggag agacttgatc tattgaaaca atatcttgaa ttaaatatta 13620 aagaagaccc tactcttaaa tatgtacaaa tat ctggatt attaattaaa tcgttcccat 136S0 caactgtaac atacgtaaga aagactgcaa tcaaatatct aaggattcgc ggtattagtc 13740 cac cgaggt aattgatgat tgggatccgg tagaagatga aaatatgctg gataacattg 13800 tcaaaactat aaatgataac tgtaataaag ataataaagg gaataaaatt aacaatttct 13860 ggggactagc acttaagaac tatcaagtcc ttaaaatcag atctataaca agtgattctg 13920 atgataatga tagactagat gctaatacaa gtggtttgac acttcctcaa ggagggaatt 13 B0 atctatcgca ttattcggaa tcaattgaga tcaacagcac tagttgtctg aaagctcttg 14040 agttatcaca aattttaatg aaggaagtca ataaagacaa ggacaggctc ttcctgggag 14100 aaggagcagg agctatgcta gcatgttatg atgccacátt aggacctgca gttaattatt 14160 tttgaatata ataattcagg acagatgtaa ttggtcaacg agaattgaaa atatttcctt 14220 cagaggtatc attagtaggt aaaaaattag gaaatgtgac acagattctt aacagggtaa 14260 aagtactgtt caatgggaat cctaattcaa catggatagg aaatatggaa tgtgagagct 14340 taatatggag tgaattaaat gataagtcca ttggattagt aeattgtgat atggaaggag 14400 atcagaagaa ctatcggtaa atgaacatta actgttctac tagtgttata agaattacat 14460 acttgattgg ggatgatgat gttgt tttag tttccaaaat tatacctaca atcactccga 14520 attggtctag aatactttat ctatataaat tatattggaa agatgtaagt ataatatcac 14580 tcaaaacttc taatcctgca teaacagaat tatatetaat ttcgaaagat gcatattgta 14640 acctagtgaa ctataatgga attgttttat caaaacttaa aagattgtca ctcttggaag 1700 aaaataatct attaaaatgg atcattttat caaagaagag gaataatgaa tggttacatc 14760 agaaggagaa atgaaatcaa agagattatg gaatcatgag accatatcat atggcactac 14820 aaatctttgg atttcaaatc aatttaaatc atctggcgaa agaattttta tcaaccccag 14880 atctgactaa tatcaacaat ataatccaaa gttttcagcg aacaataaag gatgttttat 14940 ttgaatggat taatataact agagaoataa catgatgata agatataaca attaggcgga 15000 tattcecact gaaaaataag ggaaagttaa gactgctatc gagaagacta gtattaagtt 15060 ggatttcatt atcattaccg actcgattac ttacaggtcg ctttcctgat gaaaaatttg 15120 aacatagagc acagactgga tatgtatcat tagctgatac tgatttagaa tcattaaagt 15180 tattgtcgaa aaacatcatt aagaattaca gagagtgtat aggatcaata tcatattggt 15240 agaagttaaa ttctaaccaa atacttatga aattgatcgg ttggtgccaaa ttattaggaa 15300 atataa ttcccagaca agaa cccgaagacc agttattaga aaactacaat caacatgatg 15360 aatttgatat cgattaaaac ataaatacaa tgaagatata tcctaacctt tatctttaag 15420 cctaggaata gacaaaaagt aagaaaaaca tgtaatatat atataccaaa cagagttctt 15460 ctcttgtttg gt 15492 < 210 > 61 < 211 > 15498 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Sequence of pFLC. PIV32TM, 15498 bp in sense orientation < 400 > 61 accaaacaag agaagaaact tgtctgggaa tataaattta actttaaatt aacttaggat 60 taaagacatt g & ctagaagg tcaagaaaag ggaactctat aatttcaaaa atgttgagcc 120 tatttgatac atttaatgca cgtaggcaag aaaacataac aaaatcagcc ggtggagcta 180 tcattcctgg acagaaaaat actgtctcta tattcgccct tggaccgaca ataactgatg 240 aatgacatta ataatgagaa gctcttctat ttctatctca ttcactagat aatgagaáac 300 aacatgcaca aagggcaggg ttcttggtgt ctttattgt aatggcttat gccaatccag 360 agctctacct aacaacaaat ggaagtaatg gtatgtcata cagatgtcaa tacatgattg 420 agaaagatct aaaacggcaa aagtatggag gatttgtggt taagacgaga gaga gatat 480 atgaaaagac aactgattgg atatttggaa gtgacctgga ttatgatcag gaaactatgt 540 tgcagaacgg caggaacaat tcaacaattg aagaccttgt ccacacattt gggtatccat 600 catgtttagg agctcttata atacagatct ggatagttct ggtcaaagct atcactagta 660 tctcagggtt aagaaaaggc tttttcaccc gattggaagc tttcagacaa gatggaacag 720 tgcaggcagg gctggtattg agcggtgaca gattgggtca cagtggatca atcatgcggt 780 ctcaacagag cttggtaact cttatggttg aaacattaat aacaatgaat accagcagaa 840 atgacctcac aa ccatagaa aagaatatac aaattgttgg caactacata agagatgcag 900 gtctcgcttc attcttcaat acaatcagat atggaattga gaccagaatg gcagctttga 960 ctctatccac tctcagacca gatatcaata gattaaaagc tttgatggaa ctgtatttat 1020 caaagggacc acgcgctcct ttcatctgta. tcctcagaga tcetatacat ggtgagttcg 1080 caccaggcaa ctatcetgcc atatggagct atgcaatggg ggtggcagtt gtacaaaata 1140 gagccatgca acagtatgtg acgggaagat catatetaga cattgatatg ttccagctag 1200 gacaageagt agcacgtgat gccgaagctc aaatgagctc aacactggaa gatgaacttg 1260 cgaatctaaa gagtgacaca gaaagcttga agagacatat aaggaacata aacagttcag 1320 agacatcttt ccacaaaccg acaggtggat cagecataga gatggcaata gatgaagage 1380 cgaacataga cagaacaatt gcagatcaag aacaaaatgg agaacetcaa teatecataa 1440 ctgggcagaa ttcaatatgc ggaaatagaa gcgatgatca gactgagcaa gctacagaat 1500 caagaccgaa ctgacaatat cáacaaaaca tcagagacag actaaacaag agaetcaaeg 1560 acaaagcagt acaagaagaa caaccaccca ctaatcccac aaacagaaca aaccaggacg 1620 aaatagatga tctgtttaac gcatttggaa gcaactaatc gaatcaacat tttaatctaa 1680 aataagaaaa atcaataata acttaggatt aaagaatcct atcataccgg aatatagggt 17 0 ggtaaattta gagtctgctt gaaactcaat caatagagag ttgatggaaa gcgatgctaa 1800 atcatggatt aaactatcaa cttgggaaga ggaatcaaga gataaatcaa ctaatatctc 1860 ctcggccctc aacatcattg aattcatact CAGC accgac ccccaagaag acttatcgga 1920 atcaacacaa aaacgacaca gaacccagca actcagtgcc accatctgtc aaccagaaat 1980 caaaccaaca gaaacaagtg agaaagatag tggatcaact gacaaaaata gacagtccgg 2040 gaatgtacaa gtcatcacac cagaagcaaa agatagaaat attgatcagg aaactgtaca 2100 gggagaagaa gagaggacct gcagctcaga tagtagagct gagactgtgg tctctggagg 2160 aatccccaga agcatcacag attctaaaaa tggaacccaa aacacggagg atattgatct 2220 caatgaaatt agaaagatgg ataaggactc tattgagggg aaaatgcgac aatctgcaaa 2280 tgttccaagc gagatatcag catatttaca gaagtgatga acagaacaaa gtagaaacag 2340 tgatcatgga agaagcctgg aatctatcag tacacctgat acaagatcaa taagtgttgt 2400 tactgctgca acaccagatg atgaagaaga aatactaatg ggacaaagaa aaaaatagta 2460 acacatcaag aagttcttca aagaattaaa aagatgacaa aaagggggaa aagggaaaga 2520 ecggtttaag aaatcaaaag ataccgacaa ccagatacca acatcagact acagatccac 2580 atcaaaaggg cagaagaaaa tctcaaagac aacaaccacc aacaccgaca caaaggggca 2640 cagacagaat aacagaaata catcagaaac acaatcctca tcatcatcga tcatggaatc 2700 caacaacacc gaccggaacg aacagacaag cacaactcct ccaacaacaa cttccagatc 2760 aacttataca aaagaatcga tccgaacaaa aaacccaaga ctctgaatcc cacaaaagac 2820 aaatggaaag gaaaggaagg atacagaaga gagcaatcga tttacagaga gggcaattac 2880 tctattgcag aatcttggtg taattcaatc cacatcaaaa ctagatttat atcaagacaa 2940 tgtgtagcaa acgagttgta atgtactaaa caatgtagat actgcatcaa agatagattt 3000 cctggcagga ttagtcatag gggtttcaat ggacaacgac cacagataca acaaaattaa 3060 aaatgaaatg ctaaacctca aagcagatct aaagaaaatg gacgaatcac atagaagatt 3120 gatagaaaat eaaagagaac aactgtcatt gatcacgtca ctaatttcaa atctcaaaat 3180 tatgactgag agaggaggaa agaaagacca aaatgaatcc aatgagagag tatccatgat 3240 caaaacaaaa ttgaaagaag aaaagatcaa gaagaccagg tttgacccac ttatggaggc 3300 gacaagaata acaaggcatt tacccgatct atatcgacat gcaggagata cactagagaa 3360 cgatgtacaa gttaaatcag agatattaag ttcatacaat gagtcaaatg caacaagact 3420 aaagtgagca aatacccaaa gtacaatgag atcactagtt gcagtcatca acaacagcaa 3480 tctctcacaa agcacaaaac aatcatacat aaacgaactc aaacgttgca aaaatgatga 3540 · agaagtatct gaattaatgg acatgttcaa tgaagatgtc aacaattgcc aatgatccaa 3600 acaccgaaca caaagaaacg aacagacaag aaacaacagt agatcaaaac ctgtcaacac 3660 acacaaaatc aagcagaatg aaacaacaga tatcaatcaa tatacaaata agaaaaactt 3720 aggattaaag aataaattaa tccttgtcca aaatgagtat aactaactct gcaatataca 3780 cattcccaga atcatcattc tctgaaaatg accattacca gtcatataga ctcaaagtca 3840 gaaagcagta atgaacagag ccccacatta gagttgccaa gatcggaaat ccaccaaaac 3900 acggatcccg gtatttagat gtcttcttac tcggcttctt cgagatggaa cgaatcaaag 3960 acaaatacgg gagtgtgaat gatctcgaca gtgacccgag ttacaaagtt tgtggctctg 4020 gatcatt cc aatcggattg gcta agtaca ctgggaatga ccaggaattg ttacaagccg 4080 ggatatagaa caaccaaaet gtgagaagaa cagtcaaagc gaaagagatg gttgtttaea 4140 tataaaacca cggtacaaaa gaactgtacc catggtccaa tagactaaga aaaggaatgc 4200 tgttcgatgc caacaaagtt gctcttgctc ctcaatgtct tccactagat aggagcataa 4260 aatttagagt aatcttcgtg aattgtacgg caattggatc aataaccttg ttcaaaattc 4320 ggcatcacta ctaagtcaat tctctaccca acacaatatc aatcaatctg caggtacaca 4380 taaaaacagg ggttcagact gattctaaag ggatagttca aaCtttggat gagaaaggcg 4440 aaaaatcact gaatttcatg gtccatctcg gattgatcaa aagaaaagta ggcagaatgt 4500 atactgtaaa actctgttga cagaaaatcg agaaaatgag attgatattt tctttaggac 4560 tagttggagg aatcagtctt catgtcaatg caactgggtc catatcaaaa acactagcaa 4620 gtcagctggt attcaaaaga gagatttgtt atcctttaat ggatctaaat ccgcatctca 4680 atctagttat C ggcttca tcagtagaga ttacaagagt ggatgcaact ttccaacctt 4740 ctttacctgg cgagttcaga tactatccta atattattgc aaaaggagtt gggaaaatca 4800 aacaatggaa ctagtaatct ctattttagt ccggacgtat ctattaagcc gaagcaaata 4860 aaggataatc aaaaacttag gacaaaagag gtcaatacca acaactatta gcagtcacac 4920 tcgcaagaat aagagagaag ggaccaaaaa agtcaaatag gagaaatcaa aacaaaaggt 4980 agaacaacaa acagaacace aatcaaaaca tccaactcac tcaaaacaaa aattccaaaa 5040 gagaccggca acacaacaag cactgaacat gcatcacctg catccaatga tagtatgcat 5100 ttttgttatg tacactggaa ttgtaggttc agatgccatt gctggagatc aactcctcaa 5160 tgtaggggtc attcaatcaa agataagatc actcatgtac tacactgatg gtggcgctag 5220 ctttattgtt gtaaaattac tacccaatct tcccccaagc aatggaacat gcaacatcac 5280 cagtctagat gcatataatg ttaccctatt taagttgcta acacccctga ttgagaaccc 5340 tctgctgtta gagcaaaatt cagataccaa accccgccga gaacgatttg caggagtcgt 5400 tattgggctt gctgcactag gagtagctac agctgcacaa ataaccgcag ctgtagcaat 5460 agtaaaagcc aatgcaaatg ctgctgcgat aaacaatctt gcatcttcaa ttcaatccac 5520 caacaaggca gtatccgatg tgataactgc atcaagaaca attgcaaccg cagttcaagc 5580 gattcaggat cacatcaatg gagccattgt caacgggata acatctgcat catgccgtgc 5640 ctaattgggt ccatgatgca caatattaaa tttgtatctc actgagctta ctacaatatt 5700 tcataatcaa ataacaaacc ctgcgctgac ACCAC tttcc atccaagctt taagaatcct 5760 cctcggtagc accttgccaa ttgtcattga atccaaactc aacacaaaac tcaacacagc 5820 agagctgctc agtagcggac tgttaactgg tcaaataatt tccatttccc caatgtacat 5880 attcaaatca gcaaatgcta atgttccgac atttataatg caacccggtg cgaaggtaat 5940 tgatctaatt gctatctctg caaaccataa attacaagaa gtagttgtac aagttcctaa 6000 gaatatgcaa tagaattcta atgaactaca aaactaccca gccaatgatt gtttcgtgac 6060 gtattttgta accaeactct gatacaatga gggttccccg atccctgaat cacaatatca 6120 atgcttaagg gggaatc ta attcttgcac ttttacccct attatcggga actttctcaa 6180 gcgattcgca tttgccaatg gtgtgctcta tgccaactgc aaatctttgc tatgtaagtg 6240 tgccgaccct ccccatgttg tgtctcaaga tgacaaccaa ggcatcagca taattgatat 6300 'taagaggtgc tctgagatga tgcttgacac tttttcattt aggatcacat ctacattcaa 6360 tgctacatac gtgacagact tctcaatgat taatgcaaat attgtacatc taagtcctct 6420 agacttgtca aa attcaataaa caaatca caaatctctt aaaagtgctg aggattggat 6480 tgcagatagc aacttcttcg ctaatcaagc cagaacagcc aagacacttt attcactaat 6540 ttgataatga cataattatt tcattatatt gtttataatt aatataacga taattacaat 6600 tgcaattaag tattacagaa ttcaaaagag aaatcgagtg gatcaaaatg acaagccata 6660 tgtactaaca aacaaataac atatctacag atcattagat attaaaatta taaaaaactt 6720 aggagtaaag ttacgcaatc caactctact catataattg aggaaggacc caatagacaa June 80 atccaaattc gagatggaat actggaagca taceaatcac ggaaaggatg ctggcaatga 6840 tctatggcta gctggagacg ctcatggcaa caagctcact aataagataa tatacatatt 6900 atcctggtgt atggacaata. tattatcaat agtcttcatc atagtgctaa ttaattccat 6960 ccatgagata attcatcttg atgtttcctc tggtcttatg aattctgatg agtcacagca 7020 aggcattatt cagcctatea tagaatcatt aaaatcattg attgctttgg ccaaccagat 7080 tctatataat gttgcaatag taattcctct taaaattgac agtatcgaaa ctgtaatact 7140 ctctgcttta aaagatatgc acaccgggag tatgtccaat gccaactgca cgccaggaaa 7200 tctgcttctg catgatgcag catacatcaa tggaataaac aaattccttg tacttgaatc 7260 atacaatggg acgcctaaat atggacctct cctaaatata cccagcttta tcccctcagc 7320 aacatctccc catgggtgta ctagaatacc atcattttca ctcatcaaga cccattggtg 7380 ttacactcac aatgtaatgc ttggagattg tcttgatttc acggcateta accagtattt 7440 atcaatgggg ataatacaac aatctgctgc agggtttcca attttcagga ctatgaaaac 7500 agtgatggaa catttaccta tcaatcgcaa aagctgttca gtcactgcta taccaqgagg 7560 ttgtgtcttg tattgctatg tagctacaag gtctgaaaaa gaagattatg ccacgactga 7620 tctagctgaa ctgagacttg ctttctatta ttataatgat acctttattg aaagagtcat 7680 atctcctcca aatacaacag ggcagtgggc cacaatcaac cctgcagtcg gaagcgggat ctatcatcta ggetttatct 7740 TATTT cctgt atacggtggt ctcataaatg ggactacttc 7800 ttacaatgag cagtcctcac gctattttat cccaaaacat cccaacataa cttgtgccgg 7860 taactccagc aaacaggctg caatagcacg gagttcctat gtcatccgtt atcactcaaa 7920 caggttaatt cagagtgctg ttcttatttg tccattgtct gacatgcata cagaagagtg 7980 taatctagtt atgtttaaca attcccaagt catgatgggt gcagaaggta ggctctatgt 8040 tattggtaat aatttgtatt attatcaacg cagttcctct tggtggtctg catcgctctt 8100 ttacaggatc aatacagatt tttctaaagg aattcctccg atcattgagg ctcaatgggt 8160 accgtcctat eaagttcctc gtcctggagt catgccatgc aatgcaacaa gtttttgccc 8220 tgctaattgc atcaeagggg tgtacgcaga tgtgtggccg cttaatgatc cagaactcat 8280 gtcacgtaat gctctgaacc ecaaetatcg atttgctgga gcctttctca aaaatgagtc 8340 caaccgaact aatcccacat tctacactgc atcggctaac tccctcttaa atactaccgg 8400 accaatcaca attcaacaac tacatcttca aagcagcata acctgcttta aaaacactgg 8460 aacccaaaaa atttattgtt taataataat tgaaatgggc tcatctcttt taggggagtt 8520 ccatttttaa ccaaataata gggaactaat gctttaagct tcataattaa ccataatatg 8S80 catcaatcta tctataatac aagtatatga taagtaatca gcaatcagac aatagacaaa 8640 aaaaaactta agggaaatat ggagcaaagc gtgctcggga aatggacact gaatctaaca 8700 atggcactgt atctgacata ctctatcctg agtgtcacct taactctcct atcgttaaag 8760 otaaaatagc acaattacac actattatga gtctacctca gccttatgat atggatgacg 8820 actcaatact agttatcact agacagaaaa taaaacttaa taaattggat aaaagacaac 88B0 gatctattag aagattaaaa ttaatattaa ctgaaaaagt gaatgactta ggaaaataca 8940 eatttatcag atatccagaa atgtcaaaag aaatgttcaa attatatata cctggtatta 9000 gactgaatta acagtaaagt ttacttaaag cagatagaac atatagtcaa atgactgatg 9060 gattaagaga tctatggatt aatgtgctat caaaattagc ctcaaaaaat gatggaagca 9120 attatgatct taatgaagaa attaataata tatcgaaagt tcacacaacc tataaatcag 9180 ataaatggta taatccattc aaaacatggt ttactatcaa gtatgatatg agaagattac 9240 aaaaagctcg aaatgagatc aottttaatg ttgggaagga ttataacttg ttagaagacc 9300 agaagaattt cttattgata catccagaat tggttttgat attagataaa caaaactata 9360 atggttatet aattactcct gaattagtat tgatgtattg tgacgtagte gaaggccgat 9420 ggaatataag tgcatgtgct aagttagate caaaat taca atctatgtat cagaaaggta 9480 ataacctgtg ggaagtgata gataaattgt ttccaattat gggagaaaag acatttgatg 9540 attagaacca tgatatcgtt cttgcattat ccttaattca aactcatgat cctgttaaac 9600 aactaagagg agctttttta aatcatgtgt tatccgagat tttgaatcta ggaattaata 9660 gagaatcgat taaggaattt ctgagtgtag attacattga taaaatttta gatatattta 9720 aatagatgaa ataagtctac atagcagaga ttttctcttt ttttagaaca tttgggcatc 9780 ctccattaga agctagtatt gcagcagaaa aggttagaaa atatatgtat attggaaaac 9840 aattaaaatt tgacactatt aataaatgtc atgctatctt ctgtacaata ataattaacg 9900 gatatagaga gaggcatggt ggacagtggc ctcctgtgac attacctgat catgcacacg 9960 aattcatcat aaatgcttac ggttcaaact ctgcgatatc atatgaaaat gctgttgatt 10020 attaccagag ataaaattca ctttatagga ataaattcat agagcctcag ttagatgagg 10080 atttgacaat ttatatgaaa gataaagcat tatctccaaa aaaatcaaat tgggacacag 10140 atctaattta tttatcctgc ctaacgcatc ctgtaccgta caacgaatca cgaagattag 10200 ttgaagtatt tatagcagat agtaaatttg atcctcatca gatattggat tatgtagaat 10260 ctggggactg gttagatgat ccagaattta atatttctta gaaaaagaga tagtcttaaa 10320 tcaaacagga aggtagactc tttgcaaaaa tgacatacaa aatgagagct acacaagttt 10380 cctacttgca tatcagagac aataacatag gaaaattctt tcaagaaaat gggatggcga 10440 agggagagat tgaattactt aagagattaa caaccatatc aatatcagga gttccacggt 10500 ataatgaagt gtacaataat tctaaaagec atacagatga ccttaaaacc tacaataaaa 10560 taagtaatct taatttgtct tctaatcaga aatcaaagaa atttgaattc aagtcaacgg 10620 atatctacaa tgatggatac gagactgtga gctgtttcct aacaacagat ctcaaaaaat 10680 actgtcttaa ttggagatat gaatcaacag ctctatttgg agaaacttgc aaccaaatat 10740 ttggattaaa taaattgttt aattggttac accctcgtct tgaaggaagt acaatctatg ta 10800 ggtgatcc ttactgtcct ccatcagata aagaacatat atcattagag gatcaccctg 10860 attctggttt ttacgttcat aacccaagag ggggtataga aggattttgt caaaaattat 10920 ggacactcat atctataagt gcaatacatc tagcagctgt tagaataggc gtgagggtga 10980 ctgcaatggt tcaaggagac aatcaagcta tagctgtaac cacaagagta cccaacaatt 11040 atgactacag agttaagaag gagatagttt ataaagatgt agtgagattt tttgattcat 11100 taagagaagt gatggatgat ctaggtcatg aacttaaatt aaatgaaacg attacaagca 11160 gcaagatgtt catatatagc aaaagaatct attatgatgg gagaattctt cctcaagctc 11220 taaaagcatt atctagatgt gtcttctggt cagagacagt aatagacgaa acaagatcag 11280 tttggcaaca catcttcaaa tcatttgcaa aagcaattga gaatggttat tcacctgttc 11340 taggatatgc atgctcaatt tttaagaata ttcaacaaet atatattgcc cttgggatga 21400 aactataaca atatcaatcc gagatcagta cagaatatca ttttaggaat ceaaattgga 11460 tgcaatatgc ctctttaata cctgctagtg ttgggggatt caattacatg gccacgtcaa 11520 gatgttttgt aaggaatatt ggtgatccat cagttgeegc attggctgat attaaaagat 11580 ttattaaggc gaatctatta gaccgaagCg ttetttatag gattatgaat caagaaccag 11640 gtgagtcatc ttttttggac tgggcttcag atccatattc atgcaattta ccacaatetc 11700 aaaatataac aaaaatataa caccatgata attacaa tg cagcaaggaa gattcaccaa 11760 atctggatta atccattatt ttcacaaata agaagatgaa caatgataga gaattagctg 11820 agttcctgat ggacaggaag gtaattctcc ctagagttgc acatgatatt ctagataatt 11880 ctctcacagg aattagaaat gccatagctg gaatgttaga tacgacaaaa tcactaattc 11940 gggttggcat aaatagagga ggactgacat atagtttgtt gaggaaaatc agtaattacg 12000 atatgaaaca atctagtaca ctaagtagga ctttgcgaet aattgtaagt gataaaatca 12060 agtatgaaga tatgtgttcg gtagaccttg ccatagcatt gcgacaaaag atgtggattc 12120 atttatcagg aggaaggatg ataagtggac ttgaaacgcc tgacccatta gaattactat 12180 etggggtagt aataacagga tcagaacatt gtaaaatatg ttattcttca gatggcacaa 12240 acccatatac ttggatgtat ttacccggta atatcaaaat aggatcagca gaaacaggta 12300 tatcgtcatt aagagttcct tattttggat cagtcactga tgaaagatct gaagcacaat 12360 taggatatat caagaatctt agtaaacctg caaaaqccgc aataagaata gcaatgatat 12420 atacatgggc atttggtaat gatgagatat cttggatgga agcctcacag ata gcacaaa 12480 ttttacacta cacgtgcaaa gatagtctca aaattttaac accggtagct acatcaacaa 12540 cagattaaag atttatcaca gatactgcaa attctccagt ctcagatgaa acatcattga 12600 cagattcata tcagagtcag acaatgtcca atgataacat gtctatcaaa gaagctaatg 12660 aaaccaaaga tactaatctt atttatcaac aaataatgtt aacaggatta agtgttttcg 12720 aatatttatt tagattaaaa gaaaccacag gacacaaccc tatagttatg catc-gcaca 12780 tagaagatga gtgttgtatt aaagaaagtt ttaatgatga acatattaat ccagagtcta 12840 cattagaatt aattcgatat cctgaaagta atgaatttat ttatgataaa gacccactca 12900 aagatgtgga cttatcaaaa ttaaagacca cttatggtta attgatatga ttcttacaca 12960 attattggga tgataetgac atcatacatg eaatttcaat atgtactgca attacaatag 13020 gtcacaatta cagatactat gategagata atttaaaaga gataatagtt attgcaaatg 13080 atgatgatat taatagetta atcactgaat ttttgactct tgacatactt gtatttctca 13140 agacatttgg tggattatta gtaaatcaat ttgeatacae tctttatagt ctaaaaatag 13200 aaggtaggga tctcatttgg gattatataa tgagaacact gagagatact tcccattcaa 13260 tatcaaaagt attatctaat gcattatctc atcctaaagt attca agagg ttctgggatt 13320 gtggagtttt aaaccctatt tatggtccta atactgctag tcaagaccag ataaaacttg 13380 ccctatctat atgtgaatat tcactagatc tatttatgag agaatggttg aatggtgtat 13440 cacttgaaat atacatttgt gacagcgata tggaagttgc aaatgatagg aaacaagcct 13S00 ttatttctag acacctttca tttgtttgtt gtttagcaga aattgcatct ttcggaccta 13560 acctgttaaa cttaacatac ttggagagac ttgatctatt gaaacaatat cttgaattaa 13620 atattaaaga agaccctact cttaaatatg tacaaatatc tggattatta attaaatcgt 13680 tcccatcaac tgtaacatac gtaagaaaga ctgcaateaa atatctaagg attcgcggta 13740 ttagtccacc tgaggtaatt gatgattggg atccggtaga agatgaaaat atgctggata 13800 aactataaat acattgtcaa ataaagataa gataactgta aaaattaaca taaagggaat 13860 atttctgggg actagcactt aagaactatc aagtccttaa aatcagatct ataacaagtg 13920 taatgataga attctgatga ctagatgcta atacaagtgg tttgacactt cctcaaggag 13980 ggaattatct atcgcatcaa ttgagattat tcggaatcaa cagcactagt tgtctgaaag 14040 ctcttgagtt atcacaaatt ttaatgaagg aagtcaataa agacaaggac aggctcttcc 14100 tgggagaagg agcaggagct atgctagcat gttatgatgc cac attagga cctgcagtta 14160 attattataa ttcaggtttg aatataacag atgtaattgg tcaacgagaa ttgaaaatat 14220 ggtatcatta ttccttcaga gtaggtaaaa aattaggaaa attcttaaca tgtgacacag 14280 gggtaaaagt actgttcaat gggaatccta attcaacatg gataggaaat atggaatgtg 14340 agagcttaat atggagtgaa ttaaatgata agtccattgg attagtacat tgtgatatgg 14400 cggtaaatca aaggagctat gaagaaactg ttctacatga acattatagt gttataagaa 14460 ttacatactt gáttggggat gatgatgttg ttttagtttc cctacaatca caaaattata 14520 ctccgaattg gtctagaata ctttatctat ataaattata ttggaaagat gtaagtataa 14580 tatcactcaa aacttctaat cctgcatcaa cagaattata tctaatttcg aaagatgcat 14640 attgtactat aatggaacct agtgaaattg ttttatcaaa acttaaaaga ttgtcactct 14700 taatctatta tggaagaaaa ttttatcaaa aaatggatca gaagaggaat aatgaatggt 14760 aatcaaagaa tacatcatga ggagaaagag attatggaat catgagacca tatcatatgg 14820 eactacaaat ctttggattt caaatcaatt taaatcatct ggcgaaagaa tttttatcaa 14880 ccccagatct gactaatatc aacaatataa tccaaagttt tcagcgaaca ataaaggatg 14940 ttttatttga atggattaat ataactcatg atgat aagag acataaatta ggcggaagat 15000 ataacatatt cccactgaaa aataagggaa agttaagact gctatcgaga agactagtat 15060 taagttggat ttcattatca ttatcgactc gattacttac aggtcgcttt cctgatgaaa 15120 aatttgaaca tagagcacag actggatatg tatcattagc tgatactgat ttagaateat 15180 taaagttatt gtcgaaaaac atcattaaga attacagaga gtgtatagga tcaatatcat 15240 attggtttct aaccaaagaa gttaaaatac ttatgaaatt gatcggtggt gctaaattat 15300 taggaattcc cagacaatat aaagaacccg aagaccagtt attagaaaac tacaatcaac 15360 atgatgaatt tgatatcgat taaaacataa atacaatgaa gatatatcct aacctttatc 15420 tttaagccta ggaatagaca aaaagtaaga aaaacatgta atatatatat accaaacaga 15480 gttcttctct tgtttggt 15498 < 210 > 62 < 211 > 15492 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Sequence of PFLC. PIV32C, 15474 bp in sense orientation < 400 > 62 accaaacaag agaagaaact tgtctgggaa tataaattta actttaaatt aacttaggat 60 taaagacatt gactagaagg tcaagaaaag ggaactctat aatttcaaaa atgttgagcc 120 tatttgatac atttaatgca cgtaggcaag aaaacataac aaaatcagcc ggtggagcta 180 tcattcctgg acagaaaaat actgtctcta tattcgccct tggaccgaca ataactgatg 40 aatgacatta ataatgagaa gctcttctat ttctatctca ttcactagat aatgagaaac 300 aacatgcaca aagggcaggg ttcttggtgt ctttattgtc aatggcttat gccaatccag 360 agctctacct aacaacaaat ggaagtaatg cagatgtcaa gtatgtcata tacatgattg 420 agaaagatct aaaacggcaa aagtatggag gatttgtggt taagacgaga gagatgatat 480 atgaaaagac aactgattgg atatttggaa gtgacctgga ttatgatcag gaaactatgt 540 tgcagaacgg caggaacaat tcaacaattg aagaccttgt ccacacattt gggtatccat 600 catgtttagg agctcttata atacagatct ggatagttct ggtcaaagct atcactagta 660 tctcagggtt aagaaaaggc tttttcaccc gattggaagc tttcagacaa gatggaacag 720 tgcaggcagg gctggtattg agcggtgaca gattgggtca cagtggatca atcatgcggt 780 ctcaacagag cttggtaact cttatggttg aaacattaat aacaatgaat accagcagaa 840 atgacctcac aaccata gaa aagaatatac aaattgttgg caactacata agagatgcag 900 gtctcgcttc attcttcaat acaatcagat atggaattga gaccagaatg gcagctttga 960 tctcagacca ctctatccac gatatcaata gattaaaagc tttgatggaa ctgtatttat 1020 caaagggacc acgcgctcct ttcatctgta tcctcagaga tcctatacat ggtgagttcg 1080 caccaggcaa ctatcctgcc atatggagct atgcaatggg ggtggcagtt gtacaaaata 1140 gagccatgca acagtatgtg acgggaagat catatctaga cattgatatg ttccagctag 1200 gacaagcagt agcacgtgat gccgaagctc aaatgagctc aacactggaa gatgaacttg 1260 cgaatctaaa gagtgacaca gaaagcttga agagacatat aaggaacata aacagttcag 1320 agacatcttt ccacaaaccg acaggtggat gatggcaata cagccataga gatgaagagc 1380 cgaacataga cagaacaatt gcagatcaag aacaaaatgg agaacctcaa tcatccataa 1440 ctgggcagaa ttcaatatgc ggaaatagaa gcgatgatca gactgagcaa gctacagaat 1500 caagaccgaa ctgacaatat caacaaaaca tcagagacag actaaacaag agacccaacg 1560 acaaagcagt acaagaagaa caaccaccca ctaatcccac aaacagaaca aaccaggacg 1620 aaatagatga tctgtttaac gcatttggaa gcaactaatc gaatcaacat tttaatctaa 1660 aataagaaaa atcaataata ACTT aggatt aaagaatcct atcataccgg aatatagggt 1740 ggtaaattta gagtctgctt gaaactcaat caatagagag ttgatggaaa gcgatgctaa 1800 atcatggatt aaactatcaa cttgggaaga ggaatcaaga gataaatcaa ctaatatctc 1860 ctcggccctc aacatcattg aattcatact cagcaccgac ccccaagaag acttatcgga 1920 atcaacacaa aaacgacaca gaacccagca actcagtgcc accatctgtc aaccagaaat 1980 caaaccaaca gaaacaagtg agaaagatag tggatcaact gacaaaaata gacagtccgg 2040 gaatgtacaa gtcatcacac cagaagcaaa agatagaaat attgatcagg aaactgtaca 2100 gagaggacct gggagaagaa gcagc shits tagtagagct gagactgtgg tctctggagg 2160 aatccccaga agcatcacag attctaaaaa tggaacccaa aacacggagg atattgacct 2220 caatgaaatt agaaagatgg ataaggactc tattgagggg aaaatgcgac aatctgcaaa 2280 tgttccaagc gagatatcag gaagtgatga catatttaca acagaacaaa gtagaaacag 2340 tgatcatgga agaagcctgg aatctatcag tacacctgat acaagatcaa taagrgttgt 2400 tactgctgca acaccagatg atgaagaaga aatactaatg ggacaaagaa aaaaatagta 2460 acacatcaag aagttcttca aagaattaaa aagatgacaa aaagggggaa aagggaaaga 2520 ctggtttaag aaatcaaaag ataccgacaa ccagatacca acatcagact acagatccac 2580 atcaaaaggg cagaagaaaa tctcaaagac aacaaccacc aacaccgaca caaaggggca 2640 aacagaaata cagacagaat catcagaaac acaatcctca tcatggaatc tcatcatcga 2700 caacaacacc gaccggaacg aacagacaag cacaactcct ccaacaacaa cttccagatc 2760 aaagaatcga aacttataca tccgaacaaa aaacccaaga ctctgaatcc cacaaaagac 2820 aaatggaaag gaaaggaagg atacagaaga gaqcaatcga tttacaqaga gggcaattac 2880 tctattgcag aatcttggtg taattcaatc cacatcaaaa ctagatttac atcaagacaa 2940 TGTA acgag tgtgtagcaa atgtactaaa caatgtagat actgcatcaa agatagattt 3000 ectggcagga ttagtcatag gggtttcaat ggacaacgac cacagataca acaaaattaa 3060 aaatgaaatg ctaaacctca aagcagatct aaagaaaatg gacgaatcac atagaagatt 3120 gatagaaaat caaagagaac aactgtcatt gatcacgca ctaatttcaa atctcaaaat 3130 agaggaggaa tatgactgag agaaagacca aaatgaatcc aatgagagag tatccatgat 3240 caaaacaaaa ttgaaagaag aaaagatcaa gaagaccagg tttgaeccac ttatggaggc 3300 gacaagaata acaaggcatt tacccgatct atatcgacat gcaggagata cactagagaa 3360 cgatgtacaa gttaaatcag agatattaag ttcatacaat gagtcaaatg caacaagact 3420 aaagtgagca aatacccaaa gtacaatgag atcactagtt gcagtcatca acaacagcaa 3480 tctctcacaa agcacaaaac aatcatacat aaacgaactc aaacgttgca aaaatgatga 3540 agaagtatct gaattaatgg acatgttcaa tgaagatgtc aacaattgcc aatgatccaa 3600 acaccgaaca caaagaaacg aacagacaag aaacaacagt agatcaaaac ctgtcaacac 3660 acacaaaatc aagcagaatg aaacaacaga tatcaatoaa tatacaaata agaaaaactt 3720 aggattaaag aataaattaa tccttgtcca aaatgagtat aactaactct gcaatataca 3780 cattcccaga atcatcattc tctgaaaatg gtcatataga accattacca ctcaaagtca 3840 gaaagcagta atgaacagag ccccacatta gagttgccaa gatcggaaat ccaccaaaac 3900 acggatcccg gtatttagat gtcttcttac tcggcttctt cgagatggaa cgaatcaaag 3960 acaaatacgg gagtgtgaat gatctcgaca gtgacccgag ttacaaagtt tgtggctctg 4020 gatcattacc aatcggattg gctaagtaca ctggga ATGA ccaggaattg ttacaagccg 4080 ggatatagaa caaccaaact cagtcaaagc gtgagaagaa gttgtttaca gaaagagatg 4140 cgg acaaaa tataaaacca gaactgtacc catggtccaa tagactaaga aaaggaatgc 4200 tgttegatgc caacaaagtt gctcttgctc ctcaatgtct tceactagat aggagcataa 4260 aatttagagt aatcttcgtg aattgtacgg caattggatc aataaccttg ttcaaaattc 4320 ggcatcacta ctaagtcaat tctctaccca aatcaatctg acacaatatc caggtacaca 4380 taaaaacagg ggttcagact gattctaaag ggatagttca aattttggat gagaaaggcg 4440 aaaaatcact gaatttcatg gtccateteg gattgatcaa aagaaaagta ggcagaatgt 4500 atactgtaaa actctgttga cagaaaatcg agaaaatgag attgatattt tctttaggac 4560 tagttggagg aatcagtctt catgtcaatg caactgggtc catatcaaaa acactagcaa 4620 gtcagctggt attcaaaaga gagatttgtt atcctt aat ggatctaaat ccgcatctca 4680 atctagttat ctgggcttca tc gtagaga ttacaagagt ggatgcaatt ttccaacctt 4740 ctttacctgg cgagttcaga tactatccta atattattgc aaaaggagtt gggaaaatca 4800 aacaatggaa ctagtaatct ctattttagt ccggacgtat ctattaagcc gaagcaaata 4860 aaggataatc aaaaacttag gacaaaagag gtcaatacca a caactatta gcagtcacac 4920 tcgcaagaat aagagagaag ggaccaaaaa agtcaaatag gagaaatcaa aacaaaaggt 4980 agaacaacaa acagaacacc aatcaaaaca tccaactcac tcaaaacaaa aattceaaaa 5040 gagaccggca acacaacaag cactgaacac catggatcac ctgcatccaa tgatagtatg 5100 catttttgtt atgtacactg gaattgtagg ttcagatgcc attgctggag atcaactcct 5160 caatgtaggg gtcattcaat caaagataag atcactcatg tactacactg atggtggcgc 5220 tagctttatt gttgtaaaat tactacccaa tcttccccca agcaatggaa catgcaacat 5280 gatgcatata caccagtcta atgttaccct atttaagttg ctaacacccc tgattgagaa 5340 cctgageaaa atttctgctg ttacagatac caaaccccgc cgagaacgat ttgcaggagt 5400 cgttattggg cttgctgcac taggagtagc tacagctgca caaataaccg cagctgtagc 5460 gccaatgcaa aatagtaaaa atgctgctgc gataaacaat cttgcatctt caattcaatc 5520 caccaacaag gcagtatccg atgtgataac tgcatcaaga acaattgcaa ccgcagttca 5580 agcgattcag gatcacatca atggagccat tgtcaacggg ataacatctg catcatgccg 5640 tgcccatgat gcactaattg ggtcaatatt aaatttgtat ctcactgagc ttactacaat 5"? 00 atttcataat caaataacaa accctgcgct gacaccactt tccat ccaag ctttaagaat S760 cctcetcggt agcaccttgc caattgtcat tgaatccaaa ctcaacacaa aactcaacac 5820 agcagagctg ctcagtagcg gactgttaac tggtcaaata atttccattt ccccaatgta 5880 catgcaaatg ctaattcaaa tcaatgttcc gacatttata atgcaacccg gtgcgaaggt 5940 attgctatct aattgatcta ctgcaaacca taaattacaa gaagtagttg tacaagttcc 6000 taatagaatt ctagaatatg caaatgaact acaaaactac ccagccaatg attgtttcgt 6060 gacaccaaac tctgtatttt gtagatacaa tgagggttcc ccgatccctg aatcacaata 6120 tcaatgctta agggggaatc ttaattcttg cacttttacc cctattatcg ggaactttct 6180 caagcgat c gcatttgcca atggtgtgct ctatgccaac tgcaaatctt tgctatgtaa 6240 gtgtgccgac cctccccatg ttgtgtctca agatgacaac gcataattga caaggcatca 6300 tattaagagg tgctctgaga tgatgcttga tttaggatca cactttttca catctacatt 6360 caatgctaca tacgtgacag acttctcaat gattaatgca aatattgtac atctaagtcc 6420 tctagacttg tcaaatcaaa tcaattcaat aaacaaatct cttaaaagtg ctgaggattg 6480 gattgcagat agcaacttct tcgctaatca agccagaaca gccaagacac tttattcact 6540 aag gcaatc gcattaatac tatcagtgat tactttggtt gttgtgggat tgctgattgc 6600 ctacatcatc aagCtggttt ctcaaatcca tcaattcaga gcactagctg ctacaacaat 6660 gttccacagg gagaatcctg ccgtcttttc caagaacaat catggaaaca tatatgggat 6720 atcttaggat ccctacagat cattagatat taaaattata aaaaacttag gagtaaagtt 6780 actctactca acgcaatcca tataattgag gaaggaccca atagacaaat ccaaatccat 6840 ggaagattac agcaatctat ctcttaaatc aattcctaaa aggacatgta gaatcatttt 6900 ccgaactgcc acaattcttg gcatatgcac attaattgtg gtattcttca ctatgttcaa 6960 tgagataatt catcttgatg tttcctctgg tcttatgaat tctgatgagt cacagcaagg 7020 cattattcag cctatcatag aatcattaaa atcattgatt gctttggcca accagattct 7080 atataatgtt gcaatagtaa ttcetcttaa aattgacagt atcgaaactg taatactctc 71 0 tgctttaaaa gatatgcaca ccgggagtat gtceaatgcc aactgcacgc caggaaatct 7200 gcttctgcat gatgcagcat acatcáatgg aataaacaaa ttccttgtac ttgaatcata 726a caatgggacg cctaaatatg gacctctcct aaatataccc agctttatcc cctcagcaac 7320 atctccccat gggtgtact gaataccatc attttcactc atcaagaccc attggtgtta 7380 cactcacaat gtaatgcttg gagattgtct tgatttcacg gcatctaacc agtatttatc 7440 to atggggata atacaacaat ctgctgcagg gtttccaatt ttcaggacta tgaaaaccat 7500 gatggaatca ttacctaagt atcgcaaaag ctgttcagtc actgctatac caggaggttg 7560 tgtcttgtat tgctatgtag ctacaaggtc tgaaaaagaa gattatgcca cgactgatct 7620 agctgaactg agacttgctt tctattatta taatgatacc tttattgaaa gagtcatatc 7680 tcttccaaat acaacagggc agtgggccac aatcaaccct gcagtcggaa gcgggatcta 7740 tcatctaggc tttatcttat ttcctgtata tggtggtctc ataaatggga ctacttctta 7800 caatgagcag tcctcacgct attttatccc aaaacatccc aacataactt gtgccggtaa 7660 ctccagcaaa caggctgcaa tagcacggag ttcctatgtc atccgttatc actcaaacag 7920 gttaattcag agtgctgttc ttatttgtcc attgtctgac atgcatacag aagagtgtaa 7980 tctagttatg tttaacaatt cccaagtcat gatgggtgca gaaggtaggc tctatgttat 8040 tggtaataat ttgtattatt atcaacgcag ttcctcttgg tggtctgcat cgctctttta 8100 caggatcaat acagattttt ctaaaggaat tcctccgatc attgaggctc aatgggtacc 8160 gtcctatcaa gttcctcgtc ctggagtcat gccatgcaat gcaacaagtt tttgccctgc 8220 taattgcatc acaggggtgt acgcagatgt gtggccgctt aatgatccag aactcatgtc 8280 acgtaat gct ctgaacccca actatcgatt tgctggagcc tttctcaaaa atgagtccaa 8340 ccgaactaat cccacattct acactgcatc ggctaactcc ctcttaaata ctaccggatt 8400 caacaacacc aatcacaaag cagcatatac atcttcaacc tgctttaaaa acactggaac 8460 tattgtttaa ccaaaaaatt taataattga aatgggctca tctcttttag gggagttcca 8520 aataatacca tttttaagg aactaatgct ttaagcttaa ttaaccataa tatgcatcaa 85B0 tctatctata atacaagtat atgataagta agacaataga atctgcaatc caaaagggaa 6640 atataaaaaa cttaggagca aagcgtgctc gggaaatgga aacaatggca cactgaatct 8700 etgtatctga catactctat cctgagtgtc accttaactc tcctatcgtt aaaggtaaaa 8760 tagcacaatt acacactatt atgagtctac ctcagcctta tgatatggat gacgactcaa 8820 tactagttat cactagacag aaaataaaac ttaataaatt caacgatcta ggataaaaga 8880 aaaattaata ttagaagatt aagtgaatga ttaactgaaa tacacattta cttaggaaaa 8940 agaaatgtca tcagatatcc aaagaaatgt tcaaattata tatacctggt attaacagta 9000 attattactt aagtgactga aaagcagata gaacatatag tcaaatgact gatggattaa 9060 gagatctatg gattaatgtg ctatcaaaat tagcctcaaa aaatgatgga agcaattatg 9120 atcttaatga ag aaattaat aatatatcga aagttcacac aacctataaa tcagataaafc 9180 attcaaaaca ggtataatcc tggtttacta tcaagtatga tatgagaaga ttacaaaaág 9240 gatcactttt ctcgaaatga tggga AATG aggattataa cttgttagaa gaccagaaga 9300 gatacatcca atttcttatt gaattggttt tgatattaga taaacaaaac tataatggtt 9360 atctaattac tcctgaatta gtattgatgt attgtgacgt agtcgaaggc cgatggaata 9420 taagtgcatg tgctaagtta gatccaaaat tacaatetat gtatcagaaa ggtaataacc 9480 gatagataaa tgtgggaagt ttatgggaga ttgtttccaa aaagacattt gatgtgatat 9540 cgttattaga accacttgca ttcaaactca ttatccttaa tgatcctgtt aaacaactaa 9600 gaggagcttt tttaaatcat gtgttatccg agatggaatc aatatttgaa tctagagaat 9660 atttctgagt cgattaagga gtagattaca ttgataaaat tttagatata tttaataagt 9720 tgaa ctacaataga tagca gagattttct ctttttttag aacatttggg catcctccat 9780 tatcgcagca tagaagctag gaaaatatat gaaaaggtta gtatattgga aaacaattaa 9840 aatttgaeac tattaataaa tgtcatgcta tcttctgtac aataataatt aacggatata 9900 gagagaggca tggtggacag tggcctcctg tgacattacc tgatcatgca cacgaattca 9960 tcataaatgc ttacgg ttca aactctgcga tatcatatga aaatgctgtt gattattacc 10020 agagctttat aggaataaaa ttcaataaat tcatagagcc tcagttagat gaggatttga 10080 gaaagataaa caatttatat gcattatetc caaaaaaatc aaattgggac acagtttatc 10140 Ctgcatctaa tttactgtac cgtactaacg catccaacga atcacgaaga ttagttgaag 10200 tatttatagc agatagtaaa tttgatcctc atcagatatt ggattatgta gaatctgggg 10260 tgatccagaa actggttaga tttaatattt cttatagtct taaagaaaaa gagatcaaac 10320 aggaaggtag actctttgca aaaatgacat acaaaatgag agctacacaa gttttatcag 10380 agaccctact tgcaaataac ataggaaaat tctttcaaga aaatgggatg g gaagggag 10440 agattgaatt ttaacaaeca acttaagaga tatcaatatc aggagttcca cggtataatg 10500 taattctaaa aagtgtacaa agccatacag atgaccttaa aacctacaat aaaataagta 10560 atcttaattt gtcttctaat cagaaatcaa attcaagtca agaaatttga acggatatct 10620 acaatgatgg atacgagact gtgagctgtt tcctaacaac agatctcaaa aaatactgtc 10680 ttaattggag atatgaatca acagctctat ttggagaaac ttgcaaccaa atatttggat 10740 taaataaatt gtttaattgg ttacaccctc gtcttgaagg aagtacaatc tatgtaggtg 10800 atccttactg aka tccatca gataaagaac atatatcat agaggatcac cctgattctg 10860 tcataaccca gtttttacgt agagggggta tagaaggatt ttgtcaaaaa ttatqgacac 10920 tcatatctat aagtgcaata catctagcag ctgttagaat aggcgtgagg gtgactgcaa 10980 tggttcaagg agacaatcaa gctatagctg taaccacaag agtacccaac aattatgact 11040 gaaggagata acagagttaa gtttataaag atgtagtgag attttttgat tcattaagag 11100 tgatctaggt aagtgatgga aattaaatga catgaactta aacgattata agtagcaaga 11160 tagcaaaaga tgttcatata atctattatg atgggagaat tcttcctcaa gctctaaaag 11220 cattatctag atgtgtcttc tggtcagaga cgaaacaaga cagtaataga tcagcatctt 11280 caaatttggc aacatcattt gcaaaagcaa ttgagaatgg ttattcacct gttctaggat 11340 atgcatgctc aatttttaag aatattcaac aactatatat tgcccttggg atgaatatca 11400 atccaactat aacacagaat atcagagatc agtattttag gaatccaaat tggatgcaat 11460 atgcctcttt aatacctgct agtgttgggg gatccaatta catggccatg tcaagatgtt 11520 ttgtaaggaa tattggtgat ccatcagttg ccgcattggc tgatattaaa agatttatta 11580 aggcgaatct attagaccga agtgttcttt ataggattat gaatcaagaa ccaggtgagt 11640 catctt tttt ggactgggct tcagatccat attcatgcaa tctcaaaata tttaccacaa 11700 taaccaccat gataaaaaat ataacagcaa ggaatgtatt acaagattca ccaaatccat 11760 tattatctgg attattcaca aatacaatga tgaagaatta tagaagaaga gctgagttcc 11820 tgatggacag gaaggtaatt ctccctagag ttgcacatga tattctagat aattctctca 11880 aaatgccata caggaattag gctggaatgt tagatacgac aaaatcacta attcgggttg 11940 gcataaatag eggaggactg acatatagtt tgttgaggaa aatcagtaat tacgatctag 12000 tacaatatga aacactaagt aggactttgc gaetaattgt aagtgataaa atcaagtatg 12060 aagatatgtg ttcggtagac cttgccatag cattgcgaca aaagatgtgg attcatctat 12120 caggaggaag gatgataagt ggacttgaaa cgcctgaocc attagaatta ctatctgggg 12180 tagtaataac aggatcagaa cattgtaaaa tatgttattc ttcagatggc acaaacccat 12240 atacttggat gtatttaccc ggtaatatca aaataggatc agcagaaaca ggtatatcgt 12300 cattaagagt tccttatttt ggatcagtca ctgatgaaag atctgaagca caattaggat 12360 tcttagtaaa atatcaagaa cctgeaaaag ccgcaataag aatagcaatg atatatacat 12420 gggcatttgg taatgatgag atatcttgga tggaagcctc acagatagca caaacacgtg 1248 0 caaattttac actagatagt ctcaaaattt taacaccggt agctacatca acaaatttat 12540 aaaggatact cacacagatt tgaaattctc gcaactcaga cagtacatca ttgatcagag 12600 tcagcagatt cataacaatg tccaatgata acatgtctat caaagaagct aatgaaacca 12660 aagatactaa tcttatttat caacaaataa tgttaacagg attaagtgtt ttcgaatatt 12720 tatttagatt aaaagaaacc acaggacaca accctatagt tatgcatctg cacatagaag 12780 atgag GTTG tattaaagaa agttttaatg atgaacatat taatccagag tctacattag 12840 aattaattcg atatcctgaa agtaatgaat ttatttatga taaagaccca ctcaaagatg 12900 tggacttatc aaaacttatg gttattaaag accattctta cacaattgat atgaattatt 12960 tgacatcata gggatgatac eatgcaattt tgcaattaca caatatgtac atagcagata 13020 attagatcga ctatgtcaca gataatttaa aagagataat agttattgca aatgatgatg 13080 atattaatag cttaatcact gaatttttga ctcttgacat acttgtattt ctcaagacat 13140 ttggtggatt attagtaaat caatttgeat acactcttta tagtccaaaa atagaaggta 13200 gggatctcat ttgggattat ataatgagaa cactgagaga taettcccat tcaatattaa 13260 taatgcatta aagtattatc tctcatccta aagtattcaa gaggttctgg gattgtg gag 13320 ttttaaaccc tatttatggt cctaatactg ctagtcaaga ccagataaaa cttgccctat 13380 atattcacta ctatatgtga gatctattta tgagagaatg gttgaatggt gtatcacttg 13440 aaatataoat ttgtgacagc gatatggaag ttgcaaatga taggaaacaa gcctttattt 13500 ctagacacct ttcatttgtt tgttgtttag cagaaattgc atctttcgga cctaacctgt 13560 taaacttaac atacttggag agacttgatc tattgaaaca atatcttgaa ttaaatatta 13620 aagaagaccc tactcttaaa tatgtacaaa tatctggatt attaattaaa tcgttcccat 13680 atacgtaaga caactgtaac aagactgcaa tcaaatatct aaggattcgc ggtattagtc 13740 cacctgaggt aattgatgat tgggatccgg tagaagatga aaatatgctg gataacattg 13800 tcaaaactat aaatgataac tgtaataaag ataataaagg gaataaaatt aacaatttct 13860 ggggactagc acttaagaac tatcaagtcc ttaaaatcag atctataaca agtgattctg 13920 atgataatga tagactagat gctaatacaa gtggtttgac acttcctcaa ggagggaatt 13980 tcaattgaga atctatcgca ttattcggaa tcaacagcac tagttgtctg aaagcccttg 14040 aattttaatg agttatcaca ataaagacaa aaggaagtca ggacaggctc ttcccgggag 14100 aaggagcagg agctatgcta gcatgttatg atgccacatt aggacctgca gtt aactatt 14160 ataattcagg tttgaatata acagatgtaa ttggtcaacg agaattgaaa atatttcctt 14220 cagaggtatc attagtaggt aaaaaattag gaaatgtgac acagattctt aacagggtaa 14280 aagtaccgtt caatgggaat cctaattcaa catggatagg aaatatggaa tgtgagagct 14340 táatatggag tgaattaaat gataagtcca ttggattagt acattgtgat atggaaggag 14400 atcagaagaa ctatcggtaa actgttctac atgaacatta tagtgttata agaattacat 14460 acttgattgg ggatgatgat gttgttttag tttccaaaat tatacctaea atcactccga S20 attggtctag aatactttat ctatataaat tatattggaa agatgtaagt ataatatcac 14S80 tcaaaacttc taatcctgca tcaacagaat tatatctaat ttcgaaagat gcatattgta 14640 acctagtgaa ctataatgga attgttttat caaaacttaa aagattgtca ctcttggaag 14700 aaaataatct attaaaatgg atcattttat caaagaagag gaataatgaa tggttacatc 14"i60 atgaaatcaa agaaggagaa agagattatg gaatcatgag accatatcat atggcactac 14820 aaatctttgg atttcaaatc aatttaaatc atctggcgaa agaattttta tcaaccccag 14880 atctgactaa tatcaacaat ataatccaaa gttttcagcg aacaataaag gatgttttat 14940 ttgaatggat taatataact catgatgata agagacataa attagg cgga agatataaca 15000 tattcccact gaaaaataag ggaaagttaa gactgctatc gagaagacta gtattaagtt 15060 ggatttcatt atcattatcg actcgattac ttacaggtcg ctctcccgat gaaaaatttg 15120 aacatagagc acagactgga tatgtateat tagctgatac tgatttagaa tcattaaagt 15180 tattgtcgaa aaacatcatt aagaattaca gagagtgtat aggatcaata tcatattggt 15240 agaagttaaa ttctaaccaa atacttatga aattgatcgg tggtgctaaa ttattaggaa 1S300 ttcccagaca atataaagaa cccgaagacc agttattaga aaactacaat caacatgatg 15360 aatttgatat cgattaaaac ataaatacaa tgaagatata tcctaacctt tatctttaag 15420 cctaggaata gacaaaaagt aagaaaaaca tgtaatatat atataccaaa cagagttctt 15480 ctcttgtttg gt 15492

Claims

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. An isolated infectious chimeric parainfluenza virus (PIV) comprising a nucleocapsid protein ( N) principal, a phosphoprotein (P) nucleocapsid and a large polymerase protein (L), and a genome or antigenome partial or complete PIV vector combined with one more heterologous genes or genomic segments that encode one or more antigenic determinants of one or more heterologous pathogens to form a chimeric PIV genome or antigenome. The chimeric PIV according to claim 1, wherein one or more of the heterologous genes or genomic segments encoding the antigenic determinants are added as supernumerary genes or genomic segments adjacent to or through a non-coding region of the genome or antigenome vector of Partial or complete PIV. The chimeric PIV according to claim 1, wherein one or more of the heterologous genes or genomic segments encoding the antigenic determinants are replaced by one or more of the counterpart genes or genomic segments in a partial PIV vector genome or antigenome. 4. The chimeric PIV according to claim 1, wherein one or more heterologous pathogens are a heterologous PIV and the heterologous genes or genomic segments code for one or more N, P, C, D,, M, F, HN and / or L proteins of PIV, or fragments thereof. The chimeric PIV according to claim 1, wherein the genome or antigenome vector is a partial or complete human PIV genome or HPV antigenome and the heterologous genes or genomic segments encoding the antigenic determinants are one or more of the PIV heterologous. 6. The chimeric PIV according to claim 5, wherein one or more of the heterologous PIVs are selected from HPIV1, HPIV2 or HPIV3. The chimeric PIV according to claim 5, wherein the genome or antigenome vector is a genome or antigenome of partial or complete HPIV and the heterologous genes or genomic segments that code for the antigenic determinants are from one or more of the heterologous HPIVs. 8. The chimeric PIV according to claim 7, wherein the genome or antigenome vector is a partial or complete HPIV3 genome or antigenome and the heterologous genomic segment genes encoding the antigenic determinants are from one or more of the heterologous HPIVs. 9. The chimeric PIV according to claim 8, wherein one or more of the genes or genomic segments that code for one or more antigenic determinants of HPIV1 selected from the HN and F glycoproteins of HPIV1 and the antigenic domains, fragments and epitopes of the They are added to the genome or partial or complete HPIV3 antigenome or are substituted therein. 10. The chimeric PIV according to the claim 8, wherein the genome or antigenome vector is a genome or JS antigenome of partial or complete HPIV3 and the heterologous genes or genomic segments that code for the antigenic determinants are from one or more of the heterologous HPIVs. 11. The chimeric PIV according to claim 10, wherein one or more of the genes or genomic segments that encode one or more of the antigenic determinants of HPIV1 are selected from HIV and F glycoproteins of HPIV1 and antigenic domains, fragments and epitopes of they are added to the genome or JS antigenome of partial or complete HPIV3 or are substituted therein. 12. The chimeric PIV according to the claim 9, wherein both HPIV1 genes encoding HN and F glycoproteins are substituted for the HIV and F genes of HPIV3 counterpart in a genome or HPIV3 vector antigenome. The chimeric PIV according to claim 9, wherein the chimeric genome or antigenome incorporates at least one and up to a total complement of attenuating mutations present within cp45 JS of PIV3 selected from the mutations specifying an amino acid substitution in the L protein. in a position corresponding to Tyr942, Leu992 or Thrl558 of cp45 JS; in protein N in a position corresponding to residues Val96 or Ser389 of cp45 JS, in protein C in a position corresponding to Ile96 of cp45 JS, a nucleotide substitution in the 3 'guiding sequence of the chimeric virus in a position corresponding to nucleotide 23, 24, 28 or 45 of cp45 JS and / or a mutation in a start sequence of the N gene in a position corresponding to nucleotide 62 of cp45 JS. 14. The chimeric PIV according to the claim 8, wherein one or more of the genes or genomic segments that code for one or more antigenic determinants of HPIV2 are added to or incorporated into the genome or HPIV3 antigenome or incorporated within it. The chimeric PIV according to claim 14, wherein one or more of the HPIV2 genes or genomic segments encoding one or more of the HN and / or F glycoproteins or antigenic domains, fragments or epitopes thereof are added to the genome or partial or complete HPIV3 vector antigenome or incorporated within it. 16. The chimeric PIV according to claim 6, wherein the plurality of heterologous genes or genomic segments encoding antigenic determinants of the multiple heterologous PIVs are added to or incorporated into the genome or antigenome vector of or partial HPIV. 17. The chimeric PIV according to the claim 16, wherein the plurality of heterologous genes or genomic segments encode antigenic determinants of both HPIV1 and HPIV2 is added to or incorporated into the whole or partial HPIV3 vector antigenome or genome. 18. The chimeric PIV according to the claim 17, wherein one or more of the HPIV1 genes of the genomic segments encoding one or more of the HN and / or F glycoproteins or antigenic domains, fragments or epitopes thereof and one or more of the HPIV2 genes or segments genomic encoding for one or more of the HN and / or F glycoproteins or antigenic domains, fragments or epitopes thereof are added to or incorporated into the whole or partial HPIV3 vector antigenome or genome. 19. The chimeric PIV according to the claim 18, wherein both HPIV1 genes encoding the HN and F glycoproteins are replaced by the counterpart HPIV3 HN and F genes to form a chimeric HPIV3-1 vector antigenome or genome that is further modified by the addition or incorporation of one or more genes or genomic segments that code for one or more antigenic determinants of HPIV2. 20. The chimeric PIV according to claim 19 / wherein a transcription unit comprising an open reading frame (ORF) of an HPIV2 HN gene is added to or incorporated into the chimeric HPIV3-1 vector antigenome or genome. . 21. The chimeric PIV according to claim 20, selected from PIV3r-1.2HN or PIV3r-1 cp45.2HN. 22. The chimeric PIV according to the claim 1, wherein the genome or antigenome vector is a genome or antigenome of human PIV (HPIV) partial or complete and the heterologous pathogen is selected from the measles virus, the respiratory syncytial viruses of subgroup A and subgroup B, of the mumps virus , of human papillomavirus, human type 1 and type 1 innaunodeficiency virus 2, of herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filovirus, bunyavirus, flavivirus, alphavirus and influenza viruses. The chimeric PIV according to claim 22, wherein one or more of the heterologous antigenic determinants are selected from the HA and F proteins of the measles virus, the F, G, SH and M2 proteins of the respiratory syncytial virus of subgroup A or subgroup B, the proteins HN and F of the mumps virus, the Ll protein of the human papilloma virus, the gpl60 protein of the human immunodeficiency virus type 1 or type 2, the proteins gB, gC, gD, gE, gG, gH, gl, gj, gK, gL and gM of the herpes simplex virus and cytomegalovirus, the G protein of the rabies virus, the gp350 protein of the Epstein Barr virus; the G protein of the filovirus; the G protein of the bunyavirus; the pre M, E and NSl proteins of flaviviruses and the alphavirus E protein and the antigenic domains, fragments and epitopes thereof. 24. The chimeric PIV according to claim 22, wherein the genome or antigenome vector is a genome or antigenome of partial or complete HPIV3 or a chimeric HPIV genome or antigenome comprising a genome or partial or complete HPIV3 antigenome having one or more genes or genomic segments that code for one or more antigenic determinants of the heterologous HPIV added or incorporated therein. 25. The chimeric PIV according to the claim 24, wherein the heterologous pathogen is a measles virus and the heterologous antigenic determinants are selected from the HA and F proteins of the measles virus and the antigenic domains, fragments and epitopes thereof. 26. The chimeric PIV according to the claim 25, wherein a transcription unit comprising an open reading frame (ORF) of the HA gene of the measles virus is added to or incorporated into the HPIV3 vector antigenome or genome. 27. The chimeric PIV according to the claim 26, selected from PIV3r (HA HN-L), PIV3r (HA N-P), cp45Lr (HA N-P), PIV3r (HA P.M) or cp45Lr (HA P-M). 28. The chimeric PIV according to the claim 24, wherein the genome or antigenome vector is a chimeric HPIV genome or antigenome comprising a genome or partial or complete HPIV3 antigenome having one or more genes or genomic segments encoding one or more antigenic determinants of HPIV1 added or incorporated in them. 29. The chimeric PIV according to the claim 25, wherein the heterologous pathogen is a measles virus and the heterologous antigenic determinants are selected from the HA and F proteins of the measles virus and the antigenic domains, fragments and epitopes thereof. 30. The chimeric PIV according to claim 29, wherein the transcription unit comprising an open reading arc (ORF) of an HA gene of measles virus is added to the genome or HPIV3-1 vector antigenome or is incorporated into the same that has both the ORF of F and HN of HPIV3 replaced by the ORF of HN and F of HPIV1. 31. The chimeric PIV according to claim 30, selected from PIV3r-l HAP-M or PIV3r-l HAP.M cp45L. 32. The chimeric PIV according to claim 1, wherein the genome or antigenome vector of partial or complete PIV is combined with one or more supernumerary heterologous genes or genomic segments to form the chimeric PIV genome or antigenome. 33. The chimeric PIV according to the claim 32, wherein the genome or antigenome vector is a genome or antigenome of partial or complete HPIV3 and one or more of the supernumerary heterologous genes or genomic segments are selected from HN of HPIV1, F of HPIV1, HN of HPIV2, F of HPIV2, HA of measles, and / or a translationally silent synthetic gene unit. 34. The chimeric PIV according to the claim 33, wherein one or both of the HN ORF of HPIV1 and / or HN of HPIV2 are inserted into the HPIV3 genome or antigenome vector, respectively. 35. The chimeric PIV according to the claim 33, wherein the HN ORF of HPIV1, HN of HPIV2 and HA of measles virus are inserted between the N / P, P / M and HN / L genes, respectively. 36. The chimeric PIV according to claim 33, wherein the HIV genes of HPIV1 and HN of HPIV2 are inserted between the N / P and P / M genes, respectively and a GU insert of 3918-nt is added between the HN and Y genes. L. 37. The chimeric PIV according to claim 33, which is selected from HPIV3r 1HNN-P, HPIV3r 1HNP-M, HPIV3r 2HNN-P / HPIV3r 2HNP_M, HPIV3r 1HNN_P2HNP-.M, HPIV3r 1HNN-P2HNP-M HAHN-L and HPIV3r 1HNN -P 2HNP-M 3918 38. The chimeric PIV according to claim 32, which contains one, two, three or four pathogen protective antigens. 39. The chimeric PIV according to claim 32, which contains protective antigens from one to four pathogens selected from HPIV3, HPIV1, HPIV2 and measles virus. 40. The chimeric PIV according to claim 32, wherein one or more supernumerary heterologous genes or genomic segments add a total sequence length foreign to the recombinant genome or antigenome from 30% to 50% or greater compared to the length of the HPIV3 genome type wild of 15,462 nt. 41. The chimeric PIV according to the claim 32, wherein the addition of one or more supernumerary heterologous genes or genomic segments specifies a chimeric PIV attenuation phenotype that exhibits at least a 10 to 100 fold decrease in replication in the upper and / or lower respiratory tract. 42. The chimeric PIV according to claim 1, wherein the genome or antigenome vector is a chimeric human-bovine PIV genome or antigenome. 43. The chimeric PIV according to claim 42, wherein the chimeric human-bovine vector genome or antigenome is combined with one or more heterologous genes or genomic segments that code for one or more antigenic determinants of a heterologous pathogen selected from the measles virus, of respiratory syncytial viruses of subgroup A and subgroup B, mumps virus, human papillomavirus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, human papillomavirus rabies, Epstein Barr virus, filovirus, bunyavirus, flavivirus, alphavirus and influenza virus. 44. The chimeric PIV according to claim 42, wherein the genome or antigenome vector comprises a genome or antigenome of partial or complete HPIV combined with one or more heterologous genes or genomic segments of a BPIV. 45. The chimeric PIV according to the claim 44, wherein the transcription unit comprising an open reading frame (ORF) of an N ORF of BPIV3 is replaced in the vector genome or antigenome for a corresponding N ORF of an HPIV3 vector genome. 46. The chimeric PIV according to the claim 45, wherein the genome or antigenome vector is combined with an HA gene of the measles virus as a supernumerary gene insert. 47. The chimeric PIV according to the claim 48, which is HPIV3r-NB HAP.M. 48. The chimeric PIV according to claim 42, wherein the genome or antigenome vector comprises a genome or antigenome of partial or complete BPIV combined with one or more heterologous genes or genomic segments of an HPIV. 49. The chimeric PIV according to claim 48, wherein one or more HPIV genes or genomic segments encoding the HN and / or F glycoproteins or one or more immunogenic domains, fragments or epitopes thereof are added to the genome or antigenome bovine partial or complete or are incorporated within it to form the genome or antigenome vector. 50. The chimeric PIV according to the claim 49, wherein both HPIV3 genes encoding HN and F glycoproteins are replaced by corresponding BPIV3 HN and F genes to form the genome or antigenome vector. 51. The chimeric PIV according to the claim 50, wherein the genome or antigenome vector is combined with a F or G gene of the respiratory syncytial virus (RSV) as a supernumerary gene insert. 52. The chimeric PIV according to the claim 51, which is selected from BHPIV3r-Gl or B / HPIV3r-Fl. 53. The chimeric PIV according to claim 49, wherein one or more of the HN and / or F genes of HPIV1 or genomic segments that code for one or more immunogenic domains, fragments or epitopes thereof are incorporated into the genome or partial bovine antigenome or complete to form the genome or vector antigenome, which is further modified by the incorporation of one or more of the HN and / or F genes of HPIV2 or genomic segments that code for one or more immunogenic domains, fragments or epitopes thereof for form the chimeric genome or antigenome that expresses protective antigens of both HPIV1 and HPIV2. 54. The chimeric PIV according to claim 53, which is selected from B / HPIV3. lr-2F; B / HPIV3.1r- 2HN or B / HPIV3. lr-2F, 2HN. 55. The chimeric PIV according to claim 1, wherein the vector genome or antigenome is modified to encode a chimeric glycoprotein that incorporates one or more heterologous antigenic domains fragments or epitopes of a heterologous PIV or a pathogen without PIV to form the genome or antigenome chimeric 56. The chimeric PIV according to claim 55, wherein the vector genome or antigenome is modified to encode a chimeric glycoprotein that incorporates one or more antigenic domains, fragments or epitopes of a second antigenically distinct PIV to form the chimeric genome or antigenome. 57. The chimeric PIV according to claim 55, wherein the chimeric genome or antigenome codes for a chimeric glycoprotein having antigenic domains, fragments or epitopes of two or more of the HPIV. 58. The chimeric PIV according to claim 55, wherein the heterologous genomic segment codes for a glycoprotein ectodomain that is replaced by a corresponding glycoprotein ectodomain in the genome or antigenome vector. 59. The chimeric PIV according to claim 55, wherein one or more of the heterologous genomic segments of a second antigenically distinct HPIV encoding one or more of the antigenic domains, fragments or epitopes are substituted within a genome or antigenome vector of HPIV to encode the chimeric glycoprotein. 60. The chimeric PIV according to claim 55, wherein the heterologous genomic segments encoding both a glycoprotein ectodomain and a transmembrane region are replaced by the ecto- and transmembrane glycoprotein counterparty domains in the genome or antigenome vector. 61. The chimeric PIV according to claim 55, wherein the chimeric glycoprotein is selected from the HN or F glycoproteins of HPIV. 62. The chimeric PIV according to claim 56, wherein the PIV vector genome or antigenome is a partial HPIV3 genome or antigenome and the second antigenically distinct PIV is selected from HPIV1 or HPIV2. 63. The chimeric PIV according to the claim 62, wherein the HPIV vector genome or antigenome is a partial HPIV3 genome or antigenome and the second antigenically distinct HPIV is HPIV2. 64. The chimeric PIV according to the claim 63, wherein one or more of the glycoprotein ectodomains of HPIV2 are replaced by one or more corresponding glycoprotein ectodomains in the genome or HPIV3 vector antigenome. 65. The chimeric PIV according to the claim 64, wherein both ectodomains of the HN and F glycoproteins of HPIV2 are replaced by the ectodomains of the corresponding glycoprotein HN and F in the genome or HPIV3 vector antigenome. 66. The chimeric PIV according to the claim 65, which is PIV3r-2TM. 67. The chimeric PIV according to claim 55, which is further modified to incorporate one or more and up to a complete panel of attenuating mutations identified in cp45 JS of HPIV3. 68. The chimeric PIV according to claim 55, which is PIV3r-2TMcp45. 69. The chimeric PIV according to claim 55, wherein the PIV2 ectodomain and the transmembrane regions of one or both HN and / or F glycoproteins are fused to one or more corresponding PIV3 cytoplasmic tail regions. 70. The chimeric PIV according to the claim 69, wherein the ectodomain and the transmembrane regions of both HN and F glycoproteins of PIV2 are fused to the corresponding PIV3 cytoplasmic regions HN and F. 71. The chimeric PIV according to the claim 70, which is PIV3r-2CT. 72. The chimeric PIV according to the claim 71, which is further modified to incorporate one or more and up to a complete panel of attenuating mutations identified in cp45 JS of HPIV3. 73. The chimeric PIV according to the claim 72, which is PIV3r-2CTcp45. 74. The chimeric PIV according to claim 55, which is further modified to incorporate one or more and up to a complete panel of attenuation mutations identified in cp45 JS of HPIV3 selected from the mutations specifying an amino acid substitution in protein L in a position corresponding to Tyr942, Leu992 or Thrl558 of cp45 JS; in protein N at a position corresponding to residues Val96 or Ser389 of cp45 JS, in protein C at a position corresponding to Ile96 of cp45 JS, a substitution of nucleotides in a 3 'leader sequence of the chimeric virus at a position corresponding to nucleotide 23, 24, 28 or 45 of cp45 JS and / or a mutation in the start sequence of the N gene at a position corresponding to nucleotide 62 of cp45 JS. 75. The chimeric PIV according to claim 55, wherein the plurality of heterologous genes or genomic segments encoding antigenic determinants of the multiple heterologous PIVs are added to or incorporated into the genome or antigenome vector of HPIV partial or complete. 76. The chimeric PIV according to claim 75, wherein the plurality of heterologous genes or genomic segments codes for antigenic determinants of both HPIV1 and HPIV2 and are added to incorporate a partial or complete HPIV3 vector genome or antigenome or incorporated within the same. 77. The chimeric PIV according to the claim 55, wherein the chimeric PIV genome or antigenome is attenuated by the addition or incorporation of one or more genes or genomic segments of a bovine PIV3 (BPIV3). 78. The chimeric PIV according to the claim 55, wherein the chimeric genome or antigenome is modified by the introduction of an attenuating mutation that includes an amino acid substitution of phenylalanine at position 456 of the L protein of HPIV3. 79. The chimeric PIV according to claim 78, wherein the phenylalanine at position 456 of the L protein of HPIV3 is replaced by leucine. 80. The chimeric PIV according to claim 55, wherein the chimeric genome or antigenome incorporates one or more heterologous genes or genomic segments that code for one or more antigenic determinants from the respiratory syncytial virus (RSV) or the measles virus. 81. The chimeric PIV according to claim 1, wherein the chimeric genome or antigenome is modified by the addition or substitution of one or more heterologous genes or genomic segments conferring enhanced genetic stability or altering attenuation, reactogenicity in vivo, or development in the culture of the chimeric virus. 82. The chimeric PIV according to claim 1, wherein the chimeric genome or antigenome is modified by the introduction of one or more attenuating mutations identified in a biologically derived mutant PIV or other unsegmented negative chain mutant virus. 83. The chimeric PIV according to the claim 82, wherein the chimeric genome or antigenome incorporates at least one and up to a total complement of attenuating mutations present within cp45 JS of PIV3. 84. The chimeric PIV according to claim 82, wherein the chimeric genome or antigenome incorporates at least one and up to a total complement of attenuating mutations specifying an amino acid substitution in the L protein at a position corresponding to Tyr9"2 / Leu992 or Thri558 in cp45 JS; in the N protein at a position corresponding to the Valg6 or Ser389 residues of cp45 JS, in protein C at a position corresponding to Ile96 of cp45 JS, in the F protein at a position corresponding to the residues Ile ^ oo Ala450 of cp45 JS , in the HN protein at a position corresponding to the Val38 residue of cp45 JS, a substitution of nucleotides in a 3 'leader sequence of the chimeric virus at a position corresponding to nucleotide 23, 24, 28 or 45 of cp45 JS and / or a mutation in a start sequence of the N gene at a position corresponding to nucleotide 62 of cp45 JS. 85. The chimeric PIV according to the claim 82, wherein the chimeric genome or antigenome incorporates attenuating mutations from the biologically different derived PIV or other unsegmented negative-chain RNA mutant viruses. 86. The chimeric PIV according to the claim 82, wherein the chimeric genome or antigenome incorporates an attenuating mutation at an amino acid position corresponding to an amino acid position of an attenuating mutation identified in a mutant or heterologous negative-strand RNA virus. 87. The chimeric PIV according to claim 86, wherein the attenuating mutation comprises an amino acid substitution of phenylalanine at position 456 of the L protein of HPIV3. 88. The chimeric PIV according to claim 87, wherein the phenylalanine at position 456 of the L protein of HPIV3 is replaced by leucine. 89. The chimeric PIV according to claim 82, wherein the chimeric genome or antigenome includes at least one attenuating mutation stabilized by multiple nucleotide changes at a codon specifying the mutation. 90. The chimeric PIV according to claim 1, wherein the chimeric genome or antigenome comprises an additional nucleotide modification that specifies a phenotypic change selected from a change in development characteristics, attenuation, temperature sensitivity, cold adaptation, size of plaque, restriction of the host environment or a change in immunogenicity. 91. The chimeric PIV according to claim 90, wherein the additional nucleotide modification alters one or more of the PIV N, P, C, D, V, M, F, HN and / or L genes and / or a guide 3 ', 5' tracer and / or intergenic region within the genome or antigenome vector or within heterologous genes or gene segments. 92. The chimeric PIV according to the claim 91, wherein one or more of the PIV genes is deleted in whole or in part, or the expression of the genes is reduced or removed by a mutation at an RNA editing site, by a frame shift mutation, by a mutation that alters an amino acid specified by an initiation codon, or by the introduction of one or more codons terminators in an open reading frame (ORF) of the gene. 93. The chimeric PIV according to the claim 92, wherein the additional nucleotide modification comprises a partial or complete deletion of one or more of the C, D or V ORF or one or more of the nucleotide changes that reduces or removes the expression of one or more of the C ORFs. , D or V. 9. The chimeric PIV according to claim 1, wherein the chimeric genome or antigenome is further modified to code for a cytosine. 95. The chimeric PIV according to claim 1, which incorporates a heterologous gene or genomic segment from the respiratory syncytial virus (RSV). 96. The chimeric PIV according to claim 95, wherein the heterologous gene or genomic segment encoding the F and / or G glycoproteins of RSV or the immunogenic domains, fragments, or epitopes thereof. 97. The chimeric PIV according to claim 1, which is a virus. 98. The chimeric PIV according to the claim 1, which is a subviral particle. 99. A method for stimulating the immune system of an individual to induce protection against PIV comprising administering to the individual an immunologically sufficient amount of the chimeric PIV of claim 1 combined with a physiologically acceptable carrier. 100. The method according to claim 99, wherein the chimeric PIV is administered in a dose of 103 to 107 PFU. 101. The method according to rei indication 99, wherein the chimeric PIV is administered to the upper respiratory tract. 102. The method according to claim 99, wherein the chimeric PIV is administered by spraying, droplets or aerosol. 103. The method according to claim 99, wherein the genome or vector antigenome is human PIV3 (HPIV3) and the chimeric PIV produces an immune response against HPIV1 and / or HPIV2. 104. The method according to claim 99, wherein the chimeric PIV produces a polyspecific immune response against multiple human PIV and / or against a human PIV and a pathogen without PIV. 105. The method according to claim 99, wherein the genome or antigenome vector is a genome or antigenicity of human PIV (HPIV) partial or complete and the heterologous pathogen is selected from measles virus, respiratory syncytial viruses of subgroup A and subgroup B, mumps virus, human papilloma virus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, philvirus, bunyavirus, flavivirus, alphavirus and influenza virus. 106. The method according to claim 99, wherein the chimeric PIV produces a polyspecific immune response against a human PIV virus (HPIV) and measles virus. 107. The method according to claim 106, wherein the chimeric PIV produces a polyspecific immune response against the HPIV3 and measles viruses. 108. The method according to claim 99, wherein a first, chimeric PIV according to claim 1 and a second PIV are administered sequentially or simultaneously to produce a polyspecific immune response. 109. The method according to claim 108, wherein the second PIV is a second chimeric PIV according to claim 1. 110. The method according to claim 108, wherein the first chimeric PIV and the second PIV are simultaneously administered in a mixture. 111. The method according to claim 108, wherein the first chimeric PIV and the second PIV are antigenically distinct variants of HPIV. 112. The method according to claim 111, wherein the first chimeric PIV comprises a partial or complete HPIV3 genome or antigenome combined with one or more heterologous genes or genomic segments that code for one or more antigenic determinants of a different PIV. 113. The method according to claim 111, wherein the first chimeric PIV and the second PIV each incorporate one or more heterologous genes or genomic segments that code for one or more antigenic determinants of a pathogen without PIV. 114. The method according to claim 113, wherein the first and second chimeric PIV incorporates one or more heterologous genes or genomic segments that code for one or more antigenic determinants of the same pathogen without PIV. 115. A method for sequential immunization for stimulating the immune system of an individual to induce protection against multiple pathogens comprising administering to an infant of 4 months of age an immunologically sufficient amount of a first attenuated chimeric HPIV expressing an antigenic determinant of a pathogen without PIV and one or more antigenic determinants of HPIV3 and subsequently administering an immunologically sufficient amount of a second attenuated chimeric HPIV expressing an antigenic determinant of a pathogen without PIV and one or more antigenic determinants of HPIV1 or HPIV2. 116. The method for sequential immunization according to claim 115, wherein the first attenuated chimeric HPIV is an HPIV3 that expresses an antigenic determinant of the measles virus and wherein the second chimeric chimeric HPIV is a chimeric virus of PIV3-1 which expresses a antigenic determinant of the measles virus and which incorporates one or more of the attenuating mutations of HPIV3 cp45 JS. 117. The method for sequential immunization according to claim 115, where after the first vaccination, the vaccines produce a primary antibody response against both the PIV3 pathogen and the non-PIV pathogen, but not for HPIV1 or HPIV2, and in the secondary immunization the vaccine is easily infected with the second HPIV attenuated and develops both primary antibody responses to HPIV1 or HPIV2 and a high titre secondary antibody response against the pathogen without PIV. 118. The method for sequential immunization according to claim 115, wherein the first chimeric PIV produces an immune response against HPIV3 and the second chimeric PIV produces an immune response against HPIV1 or HPIV2 and wherein both the first and the second of the chimeric PIVs produces an immune response against measles or RSV. 119. The method for sequential immunization according to claim 115, wherein the pathogen without PIV is selected from measles virus, respiratory syncytial viruses (RSV) of subgroup A and subgroup B, mumps virus, papilloma virus. human, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filovirus, bunyavirus, flavivirus, alphavirus and influenza viruses. 120. The method for sequential immunization according to claim 115, wherein the second chimeric PIV comprises a genome or antigenome vector of partial or complete HPIV3 combined with one or more genes or genomic segments that encode one or more HN and / or F glycoproteins of HPIV1 and / or HPIV2 or antigenic domains, fragments or epitopes thereof. 121. The method for sequential immunization according to claim 115, wherein the genome or antigenome partial or complete vector of the first chimeric PIV incorporates at least one and up to a total complement of attenuating mutations present within the cp45 JS of HPIV3 selected from the mutations that specify a substitution of amino acids in protein L at a position corresponding to Tyr942, Leu992 or Thrl558 of cp45 JS; in protein N at a position corresponding to residues Val96 or Ser389 of cp45 JS, in protein C at a position corresponding to Ile96 of cp45 JS, a nucleotide substitution, a 3 'guiding sequence of the chimeric virus at a position corresponding to nucleotide 23, 24, 28 or 45 of cp45 JS, and / or a mutation in the start sequence of the N gene at a position corresponding to nucleotide 62 of cp45 JS. 122. An immunogenic composition for producing an immune response against PIV comprising an immunogenically sufficient amount of the chimeric PIV according to claim 1, in a physiologically acceptable carrier. 123. The immunogenic composition according to claim 122, formulated in a dose of 103 to 107 PFU. 124. The immunogenic composition according to claim 122, formulated for administration to the upper respiratory tract by spray, drops or aerosol. 125. The immunogenic composition according to claim 122, wherein the chimeric PIV produces an immune response against one or more of the viruses selected from HPIV1, HPIV2 and HPIV3. 126. The immunogenic composition according to claim 122, wherein the chimeric PIV produces an immune response against HPIV3 and other viruses selected from HPIV1 and HPIV2. 127. The immunogenic composition according to claim 122, wherein the chimeric PIV produces a polyspecific immune response against one or more HPIV and a heterologous pathogen selected from measles virus, respiratory syncytial viruses of subgroup A and subgroup B, mumps virus, human papillomavirus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein arr virus, filovirus, bunyavirus, flavivirus, alphavirus and viruses of the influenza 128. The immunogenic composition according to claim 127, wherein the chimeric PIV produces a polyspecific immune response against HPIV3 and measles or the respiratory syncytial virus. 129. The immunogenic composition according to claim 122, further comprising a second chimeric PIV according to claim 1. 130. The immunogenic composition according to claim 129, wherein the first and second chimeric PIVs are antigenically distinct variants of HPIV and carry the same or different heterologous antigenic determinants. 131. The immunogenic composition according to claim 129, wherein the first chimeric PIV comprises a partial or complete HPIV3 genome or antigenome combined with one or more heterologous genes or genomic segments that code for one or more antigenic determinants of a heterologous pathogen without PIV . 132. The immunogenic composition according to claim 129, wherein the second chimeric PIV incorporates one or more heterologous genes or genomic segments that code for one or more antigenic determinants of the same heterologous pathogen without PIV. 133. The immunogenic composition according to claim 129, wherein the first chimeric PIV produces an immune response against HPIV3 and the second chimeric PIV produces an immune response against HPIV1 or HPIV2 and wherein the chimeric PIVs both first and second produce an immune response against the pathogen without PIV. 134. The immunogenic composition according to claim 129, wherein the heterologous pathogen is selected from measles virus, respiratory syncytial viruses of subgroup A and subgroup B (RSV), mumps virus, human papilloma viruses, immunodeficiency viruses human type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filovirus, bunyavirus, flavivirus, alphavirus and influenza viruses. 135. The immunogenic composition according to claim 129, wherein the heterologous pathogen is selected from measles virus or RSV. The immunogenic composition according to claim 129, wherein the second chimeric PIV comprises a partial HPIV3 vector genome or antigenome combined with one or more of the HPIV1 genes or genomic segments encoding one or more antigenic determinants of the HN glycoproteins and / or F of HPIV1. 137. The immunogenic composition according to claim 129, wherein the second chimeric PIV comprises a partial or complete HPIV3 vector genome or antigenome combined with one or more genes or genomic segments encoding one or more HN and / or F glycoproteins of HPIV2. or antigenic domains, fragments or epitopes thereof. 138. An isolated polynucleotide comprising a chimeric PIV genome or antigenome that includes a genome or antigenome vector of partial or complete PIV combined with one or more heterologous genes or genomic segments encoding one or more antigenic determinants of one or more heterologous pathogens for form a chimeric PIV genome or antigenome. 139. The isolated polynucleotide according to claim 138, wherein one or more heterologous genes or genomic segments encoding the antigenic determinants are aggregated adjacent to the non-coding region or within the genome or partial or complete PIV vector antigenome. 140. The isolated polynucleotide according to claim 138, wherein one or more heterologous genes or genomic segments encoding the antigenic determinants are replaced by one or more counterpart genes or genomic segments in a partial PIV vector genome or antigenome. 141. The isolated polynucleotide according to claim 138, wherein one or more of the heterologous pathogens is a heterologous PIV and the heterologous genes or genomic segments code for one or more proteins N, P, C, D, V, M, F, HN and / or L of PIV or immunogenic fragments, domains or epitopes thereof. 142. The isolated polynucleotide according to claim 138, wherein the genome or antigenome vector is a genome or antigenome of human PIV (HPIV) partial or complete and the heterologous genes or genomic segments encoding the antigenic determinants are one or more of the heterologous PIV. 143. The isolated polynucleotide according to claim 142, wherein the genome or antigenome vector is a genome or antigenome of partial or complete HPIV3 and the heterologous genes or genomic segments that code for the antigenic determinants are of HPIV1 and / or HPIV2. 144. The isolated polynucleotide according to claim 138, wherein the genome or antigenome vector is a genome or antigenome of human PIV (HPIV) partial or complete and the heterologous pathogen is selected from the measles virus, respiratory syncytial viruses of subgroup A and subgroup B, mumps virus, human papilloma virus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filovirus, bunyavirus, flavivirus, alphavirus and influenza viruses. 145. The isolated polynucleotide according to claim 144, wherein one or more of the heterologous antigenic determinants are selected from the HA and F proteins of measles virus, the F, G, SH and M2 proteins of the respiratory syncytial virus of subgroup A or subgroup B, the proteins HN and F of the virus of parotiditis, the Ll protein of the human papilloma virus, the protein gp 160 of the human immunodeficiency virus type 1 or type 2, the herpes simplex virus and the proteins gB, gC, gD, E, gG, gH, gl, gJ, gK, gL and gM of cytomegalovirus, the protein g of the rabies virus, the gp350 protein of the Epstein Barr virus; F protein of filovirus, G protein of bunyavirus, E and NS1 proteins of flavivirus and protein E of alphavirus and antigenic domains, fragments and epitopes thereof. 146. The isolated polynucleotide of claim 138, wherein the genome or antigenome vector is a genome or antigenome of partial or complete HPIV3 or a chimeric HPIV genome or antigenome comprising a partial or complete HPIV3 antigenome genome having one or more genes or genomic segments that code for one or more antigenic determinants of a heterologous HPIV added or incorporated therein. 147. The isolated polynucleotide according to claim 146, wherein the heterologous pathogen is measles virus and the heterologous antigenic determinants are selected from the HA and F proteins of measles virus and antigenic domains, fragments and epitopes thereof. 148. The isolated polynucleotide according to claim 147, wherein a transcription unit comprising an open reading frame (ORF) of an HA gene of measles virus is added to the HPIV3 genome or antigenome vector or incorporated therein. 149. The isolated polynucleotide according to claim 147, wherein the transcription unit comprising an open reading frame (ORF) of an HA gene of measles virus is added to the genome or HPIV3-1 vector antigenome or is incorporated into the same having the two ORF of HN and F of HPIV3 substituted by the ORF of HN and F of HPIV1. 150. The isolated polynucleotide according to claim 138, wherein the genome or antigenome vector of partial or complete PIV is combined with one or more of the supernumerary ether genes or genomic segments to form the chimeric PIV genome or antigenome. 151. The isolated polynucleotide according to claim 150, wherein the genome or antigenome vector is a genome or antigenome of partial or complete HPIV3 and one or more of the supernumerary heterologous genes or genomic segments are selected from HN of HPIV1, F of HPIV1, HPIV2 HN, HPIV2 F, measles HA and / or a translationally silent synthetic gene unit. 152. The isolated polynucleotide according to claim 138, wherein one, two or all of the HN ORFs of HPIV1, HN of HPIV2 and HA of measles virus are added to the genome or antigenome vector. 153. The isolated polynucleotide according to claim 138, wherein one or more of the HIV genes of HPIV1 and HN of HPIV2 and a GU insert 3918-nt are added to the genome or antigenome vector. 154. The isolated polynucleotide of claim 150, wherein one or more of the supernumerary heterologous genes or genomic segments add a total sequence length foreign to the recombinant genome or antigenome from 30% to 50% or greater compared to the length of the genome HPIV3 wild type of 15,462 nt. 155. The polynucleotide isolated according to rei indication 138, wherein the genome or antigenome vector is a chimeric human-bovine PIV genome or antigenome. 156. The isolated polynucleotide according to claim 155, wherein the chimeric human-bovine vector genome or antigenome is combined with one or more heterologous genes or genomic segments that encode one or more antigenic determinants of a heterologous pathogen selected from the measles virus , respiratory syncytial viruses of subgroup A and subgroup B, mumps virus, human papilloma virus, human immunodeficiency virus type 1 and type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus , filoviruses, bunyaviruses, flaviviruses, alphaviruses and influenza viruses. 157. The isolated polynucleotide according to claim 156, wherein the genome or antigenome vector comprises a genome or antigenome of partial or complete HPIV combined with one or more heterologous genes or genomic segments of a BPIV. 158. The isolated polynucleotide according to claim 157, wherein the transcription unit comprising an open reading frame (ORF) of an N ORF of BPIV3 is replaced in the genome or vector antigenome by a corresponding N ORF of a genome HPIV3 vector. 159. The isolated polynucleotide according to claim 158, wherein the genome or antigenome vector is combined with an HA gene of the measles virus as a supernumerary gene insert. 160. The isolated polynucleotide according to claim 138, wherein the genome or antigenome vector comprises a genome or antigenome of partial or complete BPIV combined with one or more heterologous genes or genomic segments of an HPIV. 161. The isolated polynucleotide according to claim 160, wherein one or more of the HPIV genes or genomic segments encoding HN and / or F glycoproteins or one or more immunogenic domains, fragments or epitopes thereof are added to the genome or partial or complete bovine antigenome or incorporated within it to form the genome or antigenome vector. 162. The polynucleotide isolated according to claim 161, wherein both HPIV3 genes encoding HN and F glycoproteins are replaced by the corresponding BPIV3 HN and F genes to form the genome or antigenome vector. 163. The isolated polynucleotide according to claim 162, wherein the genome or vector antigenome is combined with a F or G gene of the respiratory syncytial virus (RSV) as a supernumerary gene insert. 164. The isolated polynucleotide according to claim 138, wherein the chimeric genome or antigenome codes for a chimeric glycoprotein having antigenic domains, fragments or epitopes of a pathogen of both human PIV (HPIV) and heterologous one. 165. The isolated polynucleotide according to claim 164, wherein the chimeric genome or antigenome codes for a chimeric glycoprotein having antigenic domains, fragments or epitopes of the two or more different PIVs. 166. The isolated polynucleotide according to claim 138, wherein the chimeric genome or antigenome is modified by the introduction of one or more attenuating mutations identified in a biologically derived mutant PIV or other unsegmented negative chain mutant virus. 167. The isolated polynucleotide according to claim 138, wherein the chimeric genome or antigenome incorporates at least one and up to a total complement of attenuating mutations present within PIV3 cp45 JS. 168. The isolated polynucleotide according to claim 138, wherein the chimeric genome or antigenome incorporates an attenuating mutation of the non-segmented negative-strand heterologous RNA virus. 169. The isolated polynucleotide according to claim 138, wherein the chimeric genome or antigenome comprises an additional nucleotide modification that specifies a phenotypic change selected from a change in development characteristics, attenuation, temperature sensitivity, cold adaptation, size of plaque, restriction of the host environment or a change in immunogenicity. 170. The isolated polynucleotide according to claim 138, wherein the additional nucleotide modification alters one or more of the PIV N, P, C, D, V, M, F, HN and / or L genes and / or a region 3 'guide, 5' and / or intergenic tracer within the genome or antigenome vector or within heterologous genes or segments of the gene. 171. The isolated polynucleotide according to claim 138, wherein one or more of the genes PIV are deleted in whole or in part, or the expression of the genes is reduced or removed by a mutation at an RNA editing site, by means of a frame shift mutation, by a mutation that alters an amino acid specified by a initiation codon or by introducing one or more codons terminators in an open reading frame (ORF) of the gene. 172. A method for producing an attenuated chimeric chimeric PIV particle from one or more isolated polynucleotide molecules encoding PIV, comprising: expressing in a cell or cell-free lysate, an expression vector comprising an isolated polynucleotide comprising a genome or antigenome vector of partial or complete PIV of a human or bovine PIV combined with one or more heterologous genes or genomic segments that code for one or more antigenic determinants of one or more heterologous pathogens to form a chimeric PIV genome or antigenome the N, P and L proteins of PIV. 173. The method according to claim 172, wherein the chimeric PIV genome or antigenome and the N, P and L proteins are expressed by two or more different expression vectors. 174. An expression vector comprising a functionally linked transcriptional promoter, a polynucleotide sequence that includes a genome or antigenome vector of partial or complete PIV of a human or bovine PIV combined with one or more heterologous genes or genomic segments that encode one or more antigenic determinants of one or more heterologous pathogens to form a chimeric PIV genome or antigenome and a transcriptional terminator. 175. An isolated infectious recombinant parainfluenza virus (PIV) comprising a major nucleocapsid (N) protein, or a phosphoprotein (P) nucleocapsid, a large polymerase (L) protein, and a PIV genome or antigenome having a polynucleotide insert between 150 nucleotides (nts) and 4,000 nucleotides in length in a non-coding region < NCR) of the genome or antigenome or as a separate gene unit (GU), the polynucleotide insert that lacks a complete open reading frame (ORF) and that specifies an attenuated phenotype in the recombinant PIV. 176. The recombinant PIV according to claim 175, wherein the polynucleotide insert is introduced into the PIV genome or antigenome in a non-sense, reverse orientation, whereby the insert does not code for the protein. 177. The recombinant PIV according to claim 175, wherein the polynucleotide insert is about 2,000 nts or greater in length. 178. The recombinant PIV according to claim 175, wherein the polynucleotide insert is about 3,000 nts or greater in length. 179. The recombinant PIV according to claim 175, wherein the recombinant PIV efficiently replicates in vitro and exhibits a tenuated phenotype in vivo. RBSOMBH PB LA INVBKCIÓH Chimeric parainfluenza viruses (PIVs) are provided which incorporate a PIV vector genome or antigenome and one or more antigenic determinants of a heterologous pathogen with PIV or without PIV. These chimeric viruses are infectious and attenuated in humans and other mammals and are useful for vaccine formulations to produce immune responses against one or more of the PIV, or against a pathogen with PIV and without PIV. Also provided are isolated polynucleotide molecules and vectors that incorporate a chimeric PIV genome or antigenome that includes a partial or complete PIV vector genome or antigenome combined or integrated with one or more heterologous genes or genomic segments that encode antigenic determinants of a heterologous pathogen. with PIV or without PIV. In preferred aspects of the invention, the chimeric PIV incorporates a partial or complete human, bovine, or chimeric human-bovine PIV genome or antigenome combined with one or more heterologous genes or genomic segments from a heterologous or pathogenic PIV without PIV, wherein the chimeric virus is attenuated to be used as a vaccine agent by any of a variety of mutations and nucleotide modifications introduced into the chimeric genome or antigenome.